Novel inhibitors of NF-kappaB activation

ABSTRACT

The present invention relates to novel inhibitors of the Nuclear factor kappa B (NF-κB) activating pathway useful in the treatment of NF-κB related diseases and/or in the improvement of anti-tumor treatments. These inhibitors interfere early in the TRAF induced signaling pathway and are therefore more specific than IκB.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of co-pending application Ser.No. 09/702,953, filed Oct. 31, 2000, now U.S. Pat. No. ______, which isa continuation of International Application No. PCT/BE99/00055, filedMay 5, 1999, designating the United States of America, published inEnglish as WO 99/57133, the contents of both of which are incorporatedby this reference.

REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing appendix and acomputer readable form, both of which are hereby incorporatied byreference.

TECHNICAL FIELD

[0003] This invention relates generally to biotechnology, and moreparticularly to novel inhibitors of the Nuclear factor kappa B (NF-κB)activating pathway useful in the treatment of NF-κB related diseasesand/or in the improvement of anti-tumor treatments. The invention alsorelates to nucleic acids coding for the novel inhibitors. The inventionrelates further to the use of polypeptides, derived from theseinhibitors in the treatment of NF-κB related diseases and/or cancer.Furthermore, the invention concerns pharmaceutical preparations,comprising the novel inhibitors or the polypeptides, derived from theseinhibitors and methods of screening with these compounds.

BACKGROUND

[0004] NF-κB is an ubiquitously expressed transcription factor thatcontrols the expression of a diverse range of genes involved ininflammation, immune response, lymphoid differentiation, growth controland development. NF-κB resides in the cytoplasm as an inactive dimerconsisting of p50 and p65 subunits, bound to an inhibitory protein knownas IκB. The latter becomes phosphorylated and degraded in response tovarious environmental stimuli, such as pro-inflammatory cytokines,viruses, lipopolysaccharides, oxidants, UV light and ionizing radiation.This allows NF-κB to translocate to the nucleus where it activates genesthat play a key role in the regulation of inflammatory and immuneresponses, including genes that encode pro-inflammatory cytokines(IL-1β, TNF, GM-CSF, IL-2, IL-6, IL-11, IL-17), chemokines (IL-8,RANTES, MIP-1α, MCP-2), enzymes that generate mediators of inflammation(NO synthetase, cyclo-oxygenase), immune receptors (IL-2 receptor) andadhesion molecules (ICAM-1, VCAM-1, E-selectin). Some of these inducedproteins can in turn activate NF-κB, leading to the furtheramplification and perpetuation of the inflammatory response. Recently,NF-κB has been shown to have an anti-apoptotic role in certain celltypes, most likely by inducing the expression of anti-apoptotic genes.This function may protect tumor cells against anti-cancer treatments andopens the possibility to use NF-κB inhibiting compounds to sensitize thetumor cells and to improve the efficiency of the anti-cancer treatment.

[0005] Because of its direct role in regulating responses toinflammatory cytokines and endotoxin, activation of NF-κB plays animportant role in the development of different diseases such as (Barnesand Karin, 1997): chronic inflammatory diseases, i.e., rheumatoidarthritis, asthma and inflammatory bowel disease (Brand et al., 1996);acute diseases, i.e., septic shock (Remick, 1995); Alzheimer's diseasewhere the P-amyloid protein activates NF-κB (Behl et al., 1997);atherosclerosis, where NF-κB may be activated by oxidized lipids (Brandet al., 1997); autoimmune diseases, i.e., such as systemic lupuserythematosis (Kaltschmidt et al., 1994); or cancer by up-regulatingcertain oncogenes or by preventing apoptosis (Luque et al., 1997). Inaddition, NF-κB is also involved in viral infection since it isactivated by different viral proteins, such as occurs upon infectionwith rhinovirus, influenza virus, Epstein-Barr virus, HTLV,cytomegalovirus or adenovirus. Furthermore, several viruses such as HIVhave NF-κB binding sites in their promoter/enhancer regions (Mosialos,1997).

[0006] Because of the potential role of NF-κB in many of the abovementioned diseases, NF-κB and its regulators have drawn much interest astargets for the treatment of NF-κB related diseases. Glucocorticoids areeffective inhibitors of NF-κB, but they have endocrine and metabolicside effects when given systematically (Barnes et al., 1993).Antioxidants may represent another class of NF-κB inhibitors, butcurrently available antioxidants, such as acetyl-cysteine are relativelyweak and unspecific (Schreck et al., 1991). Aspirin and sodiumsalicylate also inhibit activation of NF-κB, but only at relatively highconcentrations (Kopp and Gosh, 1994). There are some natural inhibitorsof NF-κB such as glyotoxin, derived from Aspergillus, but thesecompounds are too toxic to be used as a drug (Pahl et al., 1996).Finally, there maybe endogenous inhibitors of NF-κB, such as IL-10, thatblocks NF-κB through an effect on IκB (Wang et al., 1995). However,total inhibition of NF-κB in all cell types for prolonged periods isunwanted, because NF-κB plays a crucial role in the immune response andother defensive responses.

[0007] An important role in the induction of NF-κB by TNF and IL1 hasrecently been demonstrated for TNF-receptor associated factors, TRAF2and TRAF6, which are recruited to the stimulated TNF-receptor and IL-1receptor, respectively (Rothe et al., 1995; Cao et al., 1996). Overexpression of TRAF2 or TRAF6 activates NF-κB, whereas dominant negativemutants inhibit TNF or IL-1 induced activation of NF-κB in most celltypes. TRAF2 knock out studies have recently shown that TRAF2 is notabsolutely required for NF-κB activation, presumably because ofredundancy within the TRAF family (Yeh et al. 1997). The TRAF inducedsignaling pathway to NF-κB was further resolved by the identification ofthe TRAF-interacting protein NIK, which mediates NF-κB activation uponTNF and IL-1 stimulation by association and activation of IκB kinase-αand β (IKK) (Malinin et al, 1997; Regnier et al., 1997; DiDonato et al.,1997; Zandi et al., 1997; Woronicz et al., 1997). The latter are part ofa large multi-protein NF-κB activation complex and are responsible forphosphorylation of IκB, leading to its subsequent degradation and totranslocation of released, active NF-κB to the nucleus. This allows amore specific inhibition of NF-κB activation by stimuli (including TNFand IL-1) that activate TRAF pathways. Based on this principle, WO97/37016 discloses the use of NIK and other TRAF interacting proteinsfor the modulation of NF-κB activity.

[0008] Another protein that can associate with TRAF2 is the zinc fingerprotein A20 (Song et al., 1996). The latter is encoded by an immediateearly response gene induced in different cell lines upon stimulation byTNF or IL-1 (Dixit et al, 1990). Interestingly, over expression of A20blocks both TNF and IL-1 induced NF-κB activation (Jaattela et al.,1996). However, the mechanism by which A20 blocks NF-κB activation istotally unknown. In contrast to NIK, A20 does not seem to act directlyon IκB resulting in alternative pathway to modulate NF-κB activation.

[0009] De Valck et al. (1997) isolated an A20 binding protein, so-called14-3-3, using a yeast two-hybrid assay and demonstrated that NF-κBinhibition was independent from the binding of A20 to 14-3-3.

SUMMARY OF THE INVENTION

[0010] It is shown herein that other new A20 interacting proteinsunexpectedly can modulate and/or inhibit NF-κB activation.

[0011] The invention includes an isolated functional protein comprisingan amino acid sequence with 70-100% homology to the amino acid sequencedepicted in SEQ ID NO:2, or comprising an amino acid sequence with70-100% homology to the amino acid sequence depicted in SEQ ID NO:19,or, in the alternative, comprising an amino acid sequence with 70-100%homology to the amino acid sequence depicted in SEQ ID NO:6.

[0012] More specifically, the functional protein comprises an amino acidsequence with 70-100% homology to the amino acids 54-647 of SEQ ID NO:2,even more specifically the functional protein comprises an amino acidsequence with 70-100% homology to the amino acids 390-647 of SEQ IDNO:2, and/or comprising an amino acid sequence with 70-100% homology tothe amino acids 420-647 of SEQ ID NO:2.

[0013] Homology, in this context, means identical or similar to thereferenced sequence, while known replacements/modifications of any ofthe amino acids provided, are included as well. A homology search inthis respect can be performed with the BLAST-P (Basic Local AlignmentSearch Tool) program well known to a person skilled in the art. For thecorresponding nucleic acid sequence homology is referred to the BLASTXand BLASTN programs known in the art.

[0014] One aspect of the invention is to offer novel modulators and/orinhibitors of TNF and/or IL-1 induced NF-κB activation pathways.

[0015] An important embodiment of the invention is a protein comprisingat least the amino acids of SEQ ID NO:2.

[0016] Another embodiment of the invention is a protein comprising atleast the amino acids 54-647 of SEQ ID NO:2, as represented in SEQ IDNO:19.

[0017] A further embodiment of the invention is a protein comprising atleast the amino acids of SEQ ID NO:6.

[0018] A further aspect of the invention is the use of a proteincomprising the amino acids 420-647 of SEQ ID NO:2 to modulate and/orinhibit the NF-κB related pathway, especially the TNF and/or IL-1induced pathways.

[0019] In addition, the invention concerns the use of a protein,comprising the consensus sequence shown in SEQ ID NO:8 and/or SEQ IDNO:9, to modulate and/or inhibit the TNF and/or IL-1 induced, NF-κBrelated pathway.

[0020] Another aspect of the invention is the use of these proteins in ascreening method to screen compounds that interfere with the interactionof these protein(s) with other protein components of the NF-κB relatedpathway.

[0021] Another embodiment of the invention is the use of the abovementioned proteins, or the use of protein components screened by theabove mentioned method to sensitize tumor cells and/or improve theanti-cancer treatment.

[0022] The present invention also relates to a method for identifyingand obtaining an activator or inhibitor of A20 interacting protein(s)comprising the steps of:

[0023] (a) combining a compound to be screened with a reaction mixturecontaining the protein of the invention and a read out system capable ofinteracting with the protein under suitable conditions;

[0024] (b) maintaining the reaction mixture in the presence of thecompound or a sample comprising a plurality of compounds underconditions which permit interaction of the protein with the read outsystem;

[0025] (c) identifying or verifying a sample and compound, respectively,which leads to suppression or activation of the read out system.

[0026] The term “read out system” in context with the present inventionmeans a DNA sequence which upon transcription and/or expression in acell, tissue or organism provides for a scorable and/or selectablephenotype. Such read out systems are well known to those skilled in theart and comprise, for example, recombinant DNA molecules and markergenes as described above.

[0027] The term “plurality of compounds” in a method of the invention isunderstood as a plurality of substances which may be identical.

[0028] The compound or plurality of compounds may be comprised in, forexample, samples, e.g., cell extracts from animals or microorganisms.Furthermore, the compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating A20 interactingproteins. The reaction mixture may be a cell free extract or maycomprise a cell or tissue culture. Suitable set ups for the method ofthe invention are known to the person skilled in the art and are, forexample, generally described in Alberts et al., Molecular Biology of theCell, (3rd ed. 1994). The plurality of compounds may be, for instance,added to the reaction mixture or culture medium, or may be injected intothe cell.

[0029] If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating A20 interactingproteins. Additionally, one can further subdivide the original sample,for example, if it consists of a plurality of different compounds, so asto reduce the number of different substances per sample. The method canthen be repeated with the subdivisions of the original sample. Dependingon the complexity of the samples, the steps described above can beperformed several times, preferably until the sample identifiedaccording to the method of the invention only comprises a limited numberof, or only one substance(s). Preferably, the sample comprisessubstances of similar chemical and/or physical properties, and mostpreferably the substances are identical. The compounds which can betested and identified according to a method of the invention may beexpression libraries, e.g., cDNA expression libraries, peptides,proteins, nucleic acids, antibodies, small organic compounds, hormones,peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995),879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198and references cited supra).

[0030] The invention also relates to a DNA sequence encoding thereferenced proteins or a DNA sequence encoding an immunologically activeand/or functional fragment of such a protein, selected from the groupconsisting of:

[0031] (a) DNA sequences comprising a nucleotide sequence encoding aprotein comprising the amino acid sequence as given in SEQ ID NO:2;

[0032] (b) DNA sequences comprising a nucleotide sequence as given inSEQ ID NO: 1;

[0033] (c) DNA sequences hybridizing with the complementary strand of aDNA sequence as defined in (a) or (b) and encoding an amino acidsequence which is at least 70% identical to the amino acid sequenceencoded by the DNA sequence of (a) or (b);

[0034] (d) DNA sequences, the nucleotide sequence of which isdegenerated as a result of the genetic code to a nucleotide sequence ofa DNA sequence as defined in any one of (a) to (c); and

[0035] (e) DNA sequences encoding a fragment of a protein encoded by aDNA sequence of any one of (a) to (d).

[0036] Thus, the invention consists of DNA molecules, also callednucleic acid sequences, encoded for the above mentioned proteinspreferably a nucleic acid sequence, with 70-100% homology to the DNAsequence depicted in SEQ ID NO:1, and/or a nucleic acid sequence with70-100% homology to the DNA sequence depicted in SEQ ID NO:5.

[0037] Homology in this context means that the respective nucleic acidmolecules or encoded proteins are functionally and/or structurallyequivalent. The nucleic acid molecules that are homologous to thenucleic acid molecules described above and that are derivatives of thenucleic acid molecules are, for example, variations of the nucleic acidmolecules which represent modifications having the same biologicalfunction. In particular, the modifications encode proteins with the sameor substantially the same biological function. They may be naturallyoccurring variations, such as sequences from other varieties or species,or mutations. These mutations may occur naturally or may be obtained bymutagenesis techniques. The allelic variations may be naturallyoccurring allelic variants as well as synthetically produced orgenetically engineered variants.

[0038] The proteins encoded by the various derivatives and variants ofthe above-described nucleic acid molecules have similar commoncharacteristics, such as biological activity, molecular weight,immunological reactivity, conformation, etc., as well as physicalproperties, such as electrophoretic mobility, chromatographic behavior,sedimentation coefficients, pH optimum, temperature optimum, stability,solubility, spectroscopic properties, etc.

[0039] The present invention also relates to vectors, particularlyplasmids, cosmids, viruses, bacteriophages and other vectors usedconventionally in genetic engineering that contain a nucleic acidmolecule according to the invention. Methods which are well known tothose skilled in the art can be used to construct various plasmids andvectors; see, for example, the techniques described in Sambrook,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory(1989) N.Y.

[0040] Alternatively, the nucleic acid molecules and vectors of theinvention can be reconstituted into liposomes for delivery to targetcells.

[0041] In a preferred embodiment, the nucleic acid molecule present inthe vector is operably linked to (a) control sequence(s) which allow theexpression of the nucleic acid molecule in prokaryotic and/or eukaryoticcells.

[0042] The term “control sequence” refers to regulatory DNA sequenceswhich are necessary to affect the expression of coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism. In procaryotes, control sequencesgenerally include promoter, ribosomal binding site, and terminators. Ineucaryotes, control sequences generally include promoters, terminatorsand, in some instances, enhancers, transactivators or transcriptionfactors. The term “control sequence” is intended to include, at aminimum, all components that are necessary for expression, and may alsoinclude additional advantageous components.

[0043] The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is known to askilled person that double-stranded nucleic acid is used.

[0044] Thus, the vector of the invention is preferably an expressionvector. An “expression vector” is a construct that can be used totransform a selected host cell and provides for expression of a codingsequence in the selected host. Expression vectors can, for instance, becloning vectors, binary vectors or integrating vectors. Expressioncomprises transcription of the nucleic acid molecule preferably into atranslatable mRNA. Regulatory elements ensuring expression inprokaryotic and/or eukaryotic cells are well known to those skilled inthe art.

[0045] The present invention furthermore relates to host cellscomprising a vector as described herein or a nucleic acid moleculeaccording to the invention wherein the nucleic acid molecule is foreignto the host cell.

[0046] By “foreign” is meant that the nucleic acid molecule is eitherheterologous or homologous with respect to the host cell. “Heterologous”means derived from a cell or organism with a different genomicbackground. “Homologous” means located in a different genomicenvironment than the naturally occurring counterpart of the nucleic acidmolecule. Thus, if the nucleic acid molecule is homologous with respectto the host cell, it is not located in its natural location in thegenome of the host cell, but it is surrounded by different genes. Inthis case, the nucleic acid molecule may be either under the control ofits own promoter or under the control of a heterologous promoter. Thevector or nucleic acid molecule, according to the invention, which ispresent in the host cell may either be integrated into the genome of thehost cell or it may be maintained in some form extra-chromosomally. Itis also possible that the nucleic acid molecule of the invention can beused to restore or create a mutant gene via homologous recombination(Paszkowski (ed.), Homologous Recombination and Gene Silencing inPlants, (Kluwer Academic Publishers 1994)).

[0047] The host cell can be any prokaryotic or eukaryotic cell, such asbacterial, insect, fungal, plant or animal cells. Preferred fungal cellsare, for example, those of the genus Saccharomyces, in particular thoseof the species S. cerevisiae.

[0048] The invention also includes a method for preparing A20interacting proteins which method comprises the cultivation of hostcells that due to the presence of a vector or a nucleic acid moleculeaccording to the invention, are able to express such a protein underconditions which allow expression of the protein and thus recovery ofthe so-produced protein from the culture.

[0049] The term “expression” means the production of a protein ornucleotide sequence in the cell. However, the term also includesexpression of the protein in a cell-free system. It includestranscription into an RNA product, post-transcriptional modificationand/or translation to a protein product or polypeptide from a DNAencoding that product, as well as possible post-translationalmodifications. Depending on the specific constructs and conditions used,the protein may be recovered from the cells, from the culture medium orfrom both. For the person skilled in the art, it is well known that itis not only possible to express a native protein, but also to expressthe protein as fusion polypeptides or to add signal sequences directingthe protein to specific compartments of the host cell, for example,ensuring secretion of the peptide into the culture medium, etc.Furthermore, such a protein and fragments thereof can be chemicallysynthesized and/or modified according to standard methods.

[0050] The terms “protein” and “polypeptide” used in this applicationare interchangeable. “Polypeptide” refers to a polymer of amino acids(amino acid sequence) and does not refer to a specific length of themolecule. Thus peptides and oligopeptides are included within thedefinition of polypeptide. This term also refers to and includespost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

[0051] The present invention furthermore relates to proteins encoded bythe nucleic acid molecules according to the invention or produced orobtained by the methods described herein, and to functional and/orimmunologically active fragments of such A20 interacting proteins. Theproteins and polypeptides of the present invention are not necessarilytranslated from a designated nucleic acid sequence. The polypeptides maybe generated in any manner, including for example, chemical synthesis,or expression of a recombinant expression system, or isolation from asuitable viral system. The polypeptides may include one or more analogsof amino acids, phosphorylated amino acids or unnatural amino acids.Methods of inserting analogs of amino acids into a sequence are known inthe art. The polypeptides may also include one or more labels, which areknown to those skilled in the art. In this context, it is alsounderstood that the proteins according to the invention may be furthermodified by conventional methods known in the art. By providing theproteins according to the present invention it is also possible todetermine fragments which retain biological activity, namely the mature,processed form. This allows the construction of chimeric proteins andpeptides comprising an amino sequence derived from the protein of theinvention, which is crucial for its binding activity. The otherfunctional amino acid sequences may be either physically linked by, forexample, chemical means to the proteins of the invention or may be fusedby recombinant DNA techniques well known in the art.

[0052] The term “functional fragment of a sequence” or “functional partof a sequence” means a truncated sequence of the original sequencereferred to. The truncated sequence (nucleic acid or protein sequence)can vary widely in length; the minimum size being a sequence ofsufficient size to provide a sequence with at least a comparablefunction and/or activity of the original sequence referred to, while themaximum size is not critical. In some applications, the maximum sizeusually is not substantially greater than that required to provide thedesired activity and/or function(s) of the original sequence. Typically,the truncated amino acid sequence will range from about 5 to about 60amino acids in length. More typically, however, the sequence will be amaximum of about 50 amino acids in length, preferably a maximum of about30 amino acids. It is desirable to select sequences of at least about10, 12 or 15 amino acids, up to a maximum of about 20 or 25 amino acids.

[0053] Furthermore, folding simulations and computer redesign ofstructural motifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski, Proteins 25 (1996), 286-299;Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computer modeling ofprotein folding can be used for the conformational and energeticanalysis of detailed peptide and protein models (Monge, J. Mol. Biol.247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).In particular, the appropriate programs can be used for theidentification of interactive sites of the inventive protein , itsreceptor, its ligand or other interacting proteins by computer assistantsearches for complementary peptide sequences (Fassina, Immunomethods 5(1994), 114-120. Further appropriate computer systems for the design ofprotein and peptides are described in the prior art, for example inBerry, Biochem. Soc. Trans. 22 (1994),1033-1036; Wodak, Ann. N. Y. Acad.Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Theresults obtained from the above-described computer analysis can be usedfor, e.g., the preparation of peptidomimetics of the protein of theinvention or fragments thereof. Such pseudopeptide analogues of thenatural amino acid sequence of the protein may very efficiently mimicthe parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224).For example, incorporation of easily available achiral amino acidresidues into a protein of the invention or a fragment thereof resultsin the substitution of amide bonds by polymethylene units of analiphatic chain, thereby providing a convenient strategy forconstructing a peptidomimetic (Banerjee, Biopolymers 39 (1996),769-777). Superactive peptidomimetic analogues of small peptide hormonesin other systems are described in the prior art (Zhang, Biochem.Biophys. Res. Commun. 224 (1996), 327-331). Appropriate peptidomimeticsof the protein of the present invention can also be identified by thesynthesis of peptidomimetic combinatorial libraries through successiveamide alkylation and testing the resulting compounds, e.g., for theirbinding and immunological properties. Methods for the generation and useof peptidomimetic combinatorial libraries are described in the priorart. See, e.g., Ostresh, Methods in Enzymology 267 (1996), 220-234;Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.

[0054] Furthermore, a three-dimensional and/or crystallographicstructure of the protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber,Bioorg. Med. Chem. 4 (1996), 1545-1558).

[0055] Furthermore, the present invention relates to antibodiesspecifically recognizing a A20 interacting protein according to theinvention or parts, i.e. specific fragments or epitopes, of such aprotein. The antibodies of the invention can be used to identify andisolate other A20 interacting proteins and genes in any organism. Theseantibodies can be monoclonal antibodies, polyclonal antibodies orsynthetic antibodies as well as fragments of antibodies, such as Fab, Fvor scFv fragments etc. Monoclonal antibodies can be prepared, forexample, by the techniques as originally described in Kohler andMilstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981),3, which comprise the fusion of mouse myeloma cells to spleen cellsderived from immunized mammals. Furthermore, antibodies or fragmentsthereof to the aforementioned peptides can be obtained by using methodswhich are described, for example, in Harlow and Lane “Antibodies, ALaboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Theseantibodies can be used, for example, for the immunoprecipitation andimmunolocalization of proteins according to the invention. Additionally,the antibodies can be used for the monitoring of the synthesis of suchproteins, for example, in recombinant organisms, and for theidentification of compounds interacting with the protein according tothe invention. For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies selections, yielding a high increment of affinity from asingle library of phage antibodies which bind to an epitope of theprotein of the invention (Schier, Human Antibodies Hybridomas 7 (1996),97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In many cases,the binding phenomena of antibodies to antigens is equivalent to otherligand/anti-ligand binding.

[0056] The invention also relates to a diagnostic composition comprisingat least one of the nucleic acid molecules, vectors, proteins,antibodies or compounds described herein and, optionally, a suitablemeans for detection.

[0057] The diagnostic compositions may be used in methods for detectingexpression of related A20 interacting proteins. This is accomplished bydetecting the presence of the corresponding mRNA which comprisesisolation of mRNA from a cell, contacting the mRNA obtained with a probecomprising a nucleic acid probe as described herein under hybridizingconditions, detecting the presence of mRNA hybridized to the probe,and/or detecting the expression of the protein in the cell. Furthermethods of detecting the presence of a protein according to the presentinvention comprises immunotechniques well known in the art, for example,enzyme-linked immunosorbent assays.

[0058] The invention also relates to a pharmaceutical compositioncomprising one or more compounds, obtained by a screening methoddescribed herein, in a biologically active amount, for the treatment ofNF-κB related diseases such as respiratory disorders, particularly adultrespiratory distress syndrome, allograft rejection, chronic inflammatorydiseases such as rheumatoid arthritis, asthma or inflammatory boweldisease, and/or autoimmune diseases such as systemic lupuserythematosis.

[0059] In another aspect, the invention relates to a pharmaceuticalcomposition comprising one or more of the A20 interacting proteins in abiologically active amount, for the treatment of NF-κB related diseasessuch as respiratory disorders, particularly adult respiratory distresssyndrome, allograft rejection, chronic inflammatory diseases such asrheumatoid arthritis, asthma or inflammatory bowel disease, and/orautoimmune diseases such as systemic lupus erythematosis.

[0060] The invention also concerns a pharmaceutical compositioncomprising one or more of the A20 interacting proteins and/or one ormore of the above mentioned compounds in a biologically active amount,for a treatment to sensitize tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061]FIG. 1: Tissue distribution of ABIN transcripts. A Northern blotof poly(A)+RNA (2 μg per lane) of various murine tissues (Clontech) wasprobed with the fragment of ABIN cloned by two hybrid analysis coveringthe C-terminal sequences ABIN (390-599). RNA size markers are indicatedin kB. Expression of β-actin served as a control for the quantity of RNAloaded.

[0062]FIG. 2: Co-immunoprecipitation of A20 and ABIN after transienttransfection of the encoding plasmids of E-tagged ABIN and GreenFluorescent Protein (GFP), GFP-A20, GFP-A20(369-775), GFP-A20(1-368) oran empty expression vector as a negative control in 239T cells.Immunoprecipitation (upper panel) was performed with anti-GFP antibodyand Western blot detection with anti E-tag antibody. To controlexpression levels of ABIN, 10 μl aliquots of lysates were separated bysodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)and Western blot detection with anti-E-tag antibody (lower panel).

[0063]FIG. 3: Co-immunoprecipitation of the C-terminal fragment of ABINlacking the putative leucine zipper structure with GFP-A20 upontransient over expression in 293T cells. Immunoprecipitation andexpression levels were detected as described for full length ABIN andare shown in the upper and lower panel, respectively.

[0064]FIG. 4 is a bar graph depicting the effect of ABIN or fragments ofABIN on the TNF- and IL-1-induced activation of NF-κB, as measured byreporter gene activity. 293T cells were transiently transfected with 100ng pUT651, 100 ng pNFconluc and 100 ng expression plasmid and stimulatedwith hTNF (1000 IU/ml) or mIL1β (20 ng/ml) during 6 hours. As a control,100 ng of plasmids encoding GFP or GFP-A20 were transfected.

[0065]FIG. 5 is a bar graph depicting the effect of transienttransfection of sub-optimal quantities of expression plasmids encodingA20 (5 ng) and ABIN (20 ng) on TNF mediated NF-κB induction in 293Tcells. In both experiments, standard deviations were less than 10%.

[0066]FIG. 6 is a bar graph depicting the effect of ABIN or fragments ofABIN on the TPA-induced activation of NF-κB, as measured by reportergene activity. 293T cells were transiently transfected with 100 ngpUT651, 100 ng pNFconluc and 100 ng expression plasmid and stimulatedwith TPA (200 ng/ml) during 6 hours. As a control, 100 ng of plasmidsencoding GFP or GFP-A20 were transfected.

[0067]FIG. 7 is a bar graph depicting the effect of ABIN2 on the TNF-and TPA-induced activation of NF-κB, as measured by reporter geneactivity. 293T cells were transiently transfected with 100 ng pUT651,100 ng pNFconluc and 600 ng expression plasmid and stimulated with hTNF(1000 IU/ml) or TPA (200 ng/ml) during 6 hours. As a control, 600 ng ofplasmid encoding GFP or GFP-A20 was transfected.

[0068]FIG. 8 is a bar graph depicting the effect of full length ABIN onNF-κB activation in 293T cells induced by over expression of TRADD, RIP,TRAF2, NIK or p65 after transfection of 300 ng of their encodingplasmids, together with 100 ng pUT651, 100 ng pNFconluc and 500 ngpCAGGS-ABIN. Cells were lysed 24 hours after transfection, andluciferase and β-galactosidase activity were measured.

[0069]FIG. 9 specifically is a bar graph depicting the effect oftruncated ABIN containing the leucine zipper structure (ABIN(390-647))on TRADD, RIP, TRAF2 or NIK induced NF-κB activation. In bothexperiments, standard deviations were less than 10%.

[0070]FIGS. 10, 11 and 12: As an overview, these figures illustrate theeffect of site specific mutations in two regions of ABIN on its bindingwith A20 and on its inhibition of NF-κB activation.

[0071]FIG. 10 shows the co-immunoprecipitation of mutated ABIN with A20after transient expression of these genes in 293T cells. Cells weretransfected with the plasmids pCAGGS-GFP or pCAGGS-GFP/A20 together withthe plasmids encoding ABIN or its site specific mutants (ABIN-MUT1,ABIN-MUT2, ABIN-MUT3 or ABIN-MUT4). Lysates of these cells wereimmunoprecipitated with polyclonal anti-GFP antibody and separated onSDS-PAGE. Western blot analysis was performed with monoclonal anti-E-tagantibody to look for the Co-immunoprecipitation of ABIN or its mutants(upper panel). Lower panels show total expression levels of GFP, GFP/A20and ABIN. In this case, a fraction of the total lysate was separated bySDS-PAGE and expression was detected with anti-GFP or anti-E-tagantibodies.

[0072]FIG. 11 is a bar graph depicting the effect of mutated ABIN onTNF-induced NF-κB activation. 293T cells were transiently transfectedwith 100 ng pUT651, 100 ng pNFconluc and 200 ng expression plasmid asindicated and stimulated with TNF (1000 IU/ml) during 6 hours. Cellextracts were analyzed for luciferase and β-galactosidase activity andplotted as luc/gal, which is representative for NF-κB activity. Eachvalue is the mean (N=3) with standard deviations less than 10%.

[0073]FIG. 12 is a bar graph depicting the dominant negative effect ofABIN-MUT2, ABIN-MUT3 and ABIN-MUT4 on the NF-κB inhibiting function ofABIN. 293T cells were transiently transfected with 100 ng pUT651, 100 ngpNFconluc and 200 ng pCAGGS-ABIN or empty vector. In addition, 600 ng ofthe expression vectors encoding ABIN-MUT2, ABIN-MUT3, ABIN-MUT4 or emptyvector were co-transfected, as indicated. Cells were stimulated with TNF(1000 IU/ml) during 6 hours. Cell extracts were analyzed for luciferaseand β-galactosidase activity and plotted as 1 uc/gal.

DETAILED DESCRIPTION OF THE INVENTION

[0074] The following definitions are provided in order to furtherillustrate and define the meaning and scope of the various terms used inthe current description.

[0075] The term “treatment”, “treating” or “treat” means any treatmentof a disease in a mammal, including: (1) preventing the disease causingthe clinical symptoms of the disease not to develop; (2) inhibiting thedisease arresting the development of the clinical symptoms; and/or (3)relieving the disease causing the regression of clinical symptoms.

[0076] The term “effective amount” means a dosage sufficient to providetreatment for the disease state being treated. This will vary dependingon the patient, the disease and the treatment being effected.

[0077] “Capable of interacting” means that a protein can form a complexwith another protein, as can be measured using a yeast two hybridsystem, or with co-immunoprecipitation, or with equivalent systems knownto people skilled in the art.

[0078] “Functional” protein or fragment means a protein or fragment thatis capable to interact with the zinc finger protein A20, or with anotherprotein of the NF-κB related pathway.

[0079] “Protein A20” (“A20”) means the TNF induced zinc finger protein,described by (Dixit et al., 1990; Opipari et al., 1990; Tewari et al.,1995), or an active fragment thereof, such as the zinc finger containingpart (amino acids 387-790 of human A20; amino acids 369-775 of murineA20).

[0080] The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”,“nucleotide sequence”, “DNA sequence” or “nucleic acid molecule(s)” asused herein refer to a polymeric form of nucleotides of any length,either ribonucleotides or deoxyribonucleotides. These terms refer onlyto the primary structure of the molecule. Thus, the term includesdouble- and single-stranded DNA, and RNA. It also includes known typesof modifications, for example, methylation, “caps” substitution of oneor more of the naturally occurring nucleotides with an analog.Preferably, the DNA sequence of the invention comprises a codingsequence encoding the above defined A20 interacting protein.

[0081] A “coding sequence” is a nucleotide sequence which is transcribedinto mRNA and/or translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to mRNA, cDNA, recombinantnucleotide sequences or genomic DNA, while introns may be present aswell under certain circumstances.

[0082] “Consensus sequence” means a stretch of at least 15 amino acids,showing 50-100% homology, preferably between 70-100% homology betweenABIN and ABIN2.

[0083] “Compound” means any chemical or biological compound, includingsimple or complex inorganic or organic molecules, peptides,peptido-mimetics, proteins, antibodies, carbohydrates or nucleic acids,that interfere in the binding between a protein depicted in SEQ IDNOS:2, 19, 6, 8 or 9 with a compound of the NF-κB related pathway, suchas A20.

[0084] As used herein, the term “composition” refers to any compositionsuch as a pharmaceutical composition comprising an active ingredient ofan isolated functional protein according to the present invention. Thismay be performed in the presence of suitable excipients known to theskilled man and may be administered in the form of any suitablecomposition as detailed below, and by any suitable method ofadministration within the knowledge of a skilled man. The preferredroute of administration is parenterally. In parenteral administration,the compositions of this invention will be formulated in a unit dosageinjectable form such as a solution, suspension or emulsion, inassociation with a pharmaceutically acceptable excipient. Suchexcipients are inherently nontoxic and non-therapeutic. Examples of suchexcipients are saline, Ringer's solution, dextrose solution and Hank'ssolution. Non-aqueous excipients such as fixed oils and ethyl oleate mayalso be used. A preferred excipient is 5% dextrose in saline. Theexcipient may contain minor amounts of additives such as substances thatenhance isotonicity and chemical stability, including buffers andpreservatives.

[0085] The isolated functional protein of the invention is administeredat a concentration that is therapeutically effective to preventallograft rejection, GVHD, allergy and autoimmune diseases. The dosageand mode of administration will depend on the individual. Generally, thecompositions are administered so that the isolated functional protein isgiven at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably,it is given as a bolus dose. Continuous short time infusion (during 30minutes) may also be used. The compositions comprising the isolatedfunctional protein according to the invention may be infused at a dosebetween 5 and 20 μg/kg/minute, more preferably between 7 and 15μg/kg/minute.

[0086] The “therapeutically effective amount” of the isolated functionalprotein needed in a specific case, according to the invention, should bedetermined as being the amount sufficient to cure the patient in need oftreatment or at least to partially arrest the disease and itscomplications. Amounts effective for such use will depend on theseverity of the disease and the general state of the patient's health.Single or multiple administrations may be required depending on thedosage and frequency as required and tolerated by the patient.

[0087] With regard to the use of the isolated functional protein of thepresent invention to prevent allograft rejection, it should be stressedthat the proteins of the present invention or the compositionscomprising the same may be administered before, during or after theorgan transplantation as is desired from case to case. In case theprotein or the compositions comprising the same are administereddirectly to the host, treatment will preferably start at the time of thetransplantation and continue afterwards in order to prevent theactivation and differentiation of host T cells against the MHC on theallograft. In case the donor organ is ex vivo perfused with thefunctional protein according to the invention or the compositionscomprising the same, treatment of the donor organ ex vivo will startbefore the time of the transplantation of the donor organ in order toprevent the activation and differentiation of host T cells against theMHC on the allograft.

[0088] The invention is hereunder further explained by way of exampleswithout being restrictive in the scope of the current invention.

EXAMPLES Example 1

[0089] Isolation of the Novel Inhibitors

[0090] The novel inhibitors of the NF-κB pathway were isolated using ayeast two-hybrid assay, with protein A20 as bait. The yeast two-hybridassay was purchased from Clontech Laboratories (Palo Alto, Calif.). Thescreening of an L929r2 cDNA library with pAS2-A20 was describedpreviously (De Valck et al., 1997). Yeast colonies expressinginteracting proteins were selected by growth on minimal media lackingTryptophan, Leucine and Histidine, in the presence of 5 mM3-amino-1,2,4-triazole and by screening for β-gal activity. Plasmid DNAwas extracted from the positive colonies and the pGAD424 vectorsencoding candidate A20 interacting proteins were recovered byelectroporation in the E. coli strain HB101 and growth on media lackingLeucine.

[0091] From 1.3×10⁶ transformants, 11 clones expressed A20 interactingproteins, including A20 itself (De Valck et al., 1996) and 14-3-3proteins (De Valck et al., 1997). Three clones contained C-terminalfragments of the same cDNA encoding an unknown protein that herewith isnamed A20 Binding Inhibitor of NF-κB activation (ABIN) and 1 clonecontained the C-terminal fragment (1136 bp) of an unknown protein thatherewith is called ABIN2.

[0092] Full length ABIN cDNA was subsequently isolated from the L929r2cDNA library by colony hybridization (De Valck et al.,1996) with an ABINfragment (corresponding to amino acids 390-599) cloned by two-hybridanalysis as a probe. Several cDNA's were isolated and in the longestcDNA stop codons were identified in all three reading frames 5′ of apotential initiator methionine. Two different splice variants were foundof approximately 2800 and 2600 nucleotides long, with an open readingframe of 1941 and 1781 nucleotides respectively, initiating at twodifferent methionines (ABIN (1-647) (SEQ ID NO:2) and ABIN (54-647) (SEQID NO:19)). These cDNA's encode proteins of 72 and 68 kDa containing anamphipathic helix with 4 consecutive repeats of a leucine followed by 6random amino acids residues characteristic of a leucine zipperstructure.

[0093] Full length cDNA of ABIN2 was isolated from murine heart by 5′RACE (SMART PCR cDNA synthesis kit, Clontech), using a 3′ primerhybridizing to an EST clone (572231) which corresponds to the ABIN2fragment isolated by two hybrid analysis, but with 507 extra nucleotidesat the 5′ end. A 1,967 nucleotide long cDNA was isolated (SEQ ID NO:5),with an open reading frame of 1,290 nucleotides long, encoding a proteinof 430 amino acids (SEQ ID NO:6).

Example 2

[0094] Expression Pattern of ABIN and ABIN2.

[0095] Northern blot analysis revealed that both ABIN and ABIN2 areexpressed in all murine tissues tested (heart, brain, spleen, lung,liver, skeletal muscle, kidney, testis: see FIG. 1; only the data forABIN are shown). ABIN is present as mRNA of approximately 2800 bp whichis in accordance with the length of the cloned full length cDNA. Incontrast to A20, ABIN mRNA is constitutively expressed in bothTNF-sensitive and TNF-resistant subclones derived from the parental cellline L929s, irrespective of TNF stimulation.

[0096] ABIN2 is present as mRNA of approximately 2,000 bp, which is inaccordance with the length of the cloned, full length cDNA.

Example 3

[0097] Study of the Interaction of the ABIN and ABIN2 Proteins andProtein Fragments With A20.

[0098] Full length ABIN(1-647) and ABIN(54-647) were able to bind A20 ina yeast two hybrid assay, confirming the original interaction found withthe 3 C-terminal fragments ABIN(390-599), ABIN(249-647) andABIN(312-647). The latter contain the putative leucine zipper proteininteraction motif (397-420).

[0099] Further analysis was carried out by co-immunoprecipitation. Theeukaryotic plasmids for ABIN and its fragments as well as for ABIN2 weremade by inserting the corresponding PCR fragment in frame with anN-terminal E-tag into the mammalian expression plasmid pCAGGS (Niwa etal., 1991). cDNA encoding mutant GFP(S65T) and a fusion protein ofGFP(S65T) with murine A20 were also cloned in pCAGGS.

[0100] 2×10⁶ human embryonic kidney 293T cells were plated on 10-cmPetri dishes and transiently transfected with the suitable plasmids bycalcium phosphate-DNA co-precipitation. 24 hours after transfection,cells were lysed in 500 μl of lysis buffer (50 mM Hepes pH 7.6, 250 mMNaCl, 0.1% Nonidet P-40 and 5 mM EDTA). Lysates were incubated with 5 μlof rabbit anti-GFP antibody (Clontech) and immunocomplexes wereimmobilized on protein A-trisacryl (Pierce). The latter was washed twicewith lysis buffer and twice with lysis buffer containing 1 M NaCl.Co-precipitating proteins were separated by SDS-PAGE and revealed byWestern blotting with mouse anti-E-tag antibody (Pharmacia).

[0101] Full length ABIN as well as the C-terminal fragment lacking theleucine zipper motif (ABIN(420-647)) was still able toco-immunoprecipitate with A20 in 293T cells that were transientlytransfected with an expression plasmid for chimeric GFP-A20 protein andfull length or truncated ABIN with an N-terminal E-tag (FIG. 2).Interaction of ABIN with A20 required the C-terminal, zinc fingercontaining part of A20 (A20(369-775)). This domain was shown previouslyto be required for dimerization of A20 and for the interaction of A20with 14-3-3 protein (De Valck et al., 1996; De Valck et al., 1997). Incontrast, the N-terminal part of human A20 (A20(1-386)) was previouslyshown to interact with TRAF2 (Song et al., 1996), suggesting that A20acts as an adapter protein between TRAF2 and ABIN. The interactionbetween A20 and ABIN was not influenced by stimulation with TNF.

[0102] To characterize the sub-cellular distribution of ABIN, wetransiently transfected GFP-A20- and E-tagged ABIN cDNA in 293T cellsand analyzed their expression by means of GFP fluorescence andimmunofluorescence via the anti-E tag antibody. 4×10⁵ 293T cells wereseeded on cover slips in 6-well plates and transfected with 1 μg plasmidDNA. 24 hours after transfection, cells were fixed on the cover slipswith 3% paraformaldehyde. Upon permeabilization with 1% Triton X-100,the cells were incubated for 2 hours with mouse anti-E-tag antibody({fraction (1/1000)}) followed by a second incubation with anti-mouse Igantibody coupled to biotin (Amersham, {fraction (1/1000)}). Aftersubsequent incubation with streptavidin coupled to Texas Red (Amersham),fluorescence can be analyzed via fluorescence microscopy (Zeiss,Axiophot) using a filter set with excitation at 543 nm and emission at600 nm. In the same cells, fluorescence of GFP can be detected at adifferent wave length, namely absorption at 485 nm and emission at 510nm.

[0103] ABIN co-localized with A20 throughout the cytoplasm, both inunstimulated and in TNF stimulated cells. This observation makes theexistence of regulatory redistribution events rather unlikely.

Example 4

[0104] Sequence Analysis of the cDNA's.

[0105] Nucleotide sequence analysis was carried out using cyclesequencing on an ABI373A sequencer (Applied Biosystems, Foster City,Calif.). The sequence of full length ABIN is shown in SEQ ID NO:1; thesequence of ABIN2 is shown in SEQ ID NO:5.

[0106] Database similarity searches (BLAST) showed that ABIN is themurine homologue of the partial human cDNA encoding a protein with anunknown function (Genbank accession number D30755; Nagase et al., 1995).Moreover, ABIN shows homology with a partial human immunodeficiencyvirus (HIV) Nef interacting protein, NIP40-1 (no called Naf1=nefassociated factor 1; Fukushi et al., FEBS Letters, 442 (1999), 83-88).HIV-Nef contributes substantially to disease pathogenesis by augmentingvirus replication and markedly perturbing T-cell function.Interestingly, the effect of Nef on host cell activation has beenexplained in part by its interaction with specific cellular proteinsinvolved in signal transduction (Harris, 1996) of which ABIN might be anexample.

[0107] There are no proteins in the database that are clearly homologouswith ABIN2. However, by comparing ABIN2 with ABIN, one can define twohomologous regions and derive two consensus sequences (SEQ ID NO:8 andSEQ ID NO:9)) that may be important for the interaction of theseproteins with A20, and/or for their further function in signaltransduction. A consensus sequence of ABIN and ABIN2 is found from aminoacids 423 to 441 and 475 to 495 of SEQ ID NO:2 for ABIN, and from aminoacids 256 to 274 and 300 to 320 of SEQ ID NO:6 for ABIN2. The consensussequence illustrates 22 amino acids from ABIN and ABIN2 that overlapwith one another.

Example 5

[0108] Role of ABIN, ABIN Fragments and/or ABIN2 in the TNF-, IL-1-and/or TPA-induced Transduction Pathway Leading to NF-κB Activation, asMeasured by Reporter Gene Activity.

[0109] The construction of the ABIN, ABIN-fragments and ABIN2 plasmidswas carried out as described above. The plasmid pNFconluc, encoding aluciferase reporter gene driven by a minimal NF-κB responsive promoterdescribed by Kimura et al. (1986) and the plasmid pUT651, encodingβ-galactosidase was obtained from Eurogentec (Seraing, Belgium). NF-κBactivity was determined by the NF-κB dependent expression of aluciferase reporter gene. Therefore, 293T cells were plated in 6-wellplates at 4×10⁵ cells per well and transiently transfected by thecalcium phosphate-DNA coprecipitation method. Each transfectioncontained 800 ng of the expression plasmids, as well as 100 ng ofpNFconluc plasmid as reporter and 100 ng pUT651 plasmid as a referencefor transfection efficiency. 24 hours after transfection, these cellswere trypsinized and seeded on a 24-well plate. Another 24 hours later,cells were either stimulated with 1000 IU/ml hTNF, 20 ng/ml mIL1-β, 200ng/ml TPA (Sigma) or left untreated. After 6 hours of stimulation, cellswere lysed in 200 μl lysis buffer and analyzed for luciferase andβ-galactosidase activity as described in De Valck et al., 1997. GFP andGFP-A20 served as negative and positive controls, respectively.

[0110] Similar to A20, both splice variants of ABIN were able to blockTNF or IL-1 induced NF-κB activation in these cells, with the shorterN-terminal truncated isoform being slightly more effective (FIG. 4).Moreover, structure-function analysis of ABIN deletion mutants revealedthat the NF-κB inhibiting activity resides in the C-terminal 228 aminoacids (ABIN(420-647) SEQ ID NO:10) which are also sufficient forinteraction with A20. The latter ABIN-mutant no longer contains theleucine zipper structure, demonstrating that this protein domain is notinvolved in the interaction with A20 nor in the inhibition of NF-κB(FIG. 4). Overexpression of a combination of suboptimal doses of A20 andABIN, that on their own were not sufficient to inhibit NF-κB activation,diminished NF-κB activation upon stimulation with TNF (FIG. 5) or IL-1considerably. This suggests that ABIN mediates the NF-κB inhibitingeffect of A20.

[0111] Total ABIN (1-647; SEQ ID NO:2), the shorter splice variant(54-647; SEQ ID NO:19) and the C-terminal fragment (390-647) are alsoable to block the TPA induced NF-κB activation (FIG. 6).

[0112] Similar results were obtained when ABIN2 was used in the testinstead of ABIN or ABIN-fragments (FIG. 7).

Example 6

[0113] Effect of ABIN and/or ABIN Fragments on NF-κB Activation Inducedby Overexpression of TRADD, RIP, TRAF2, NIK or p65.

[0114] Expression vectors for ABIN and ABIN fragments were constructedas described above. The expression vectors containing TRAF2, NIK and p65have previously been described (Malinin et al., 1997; Rothe et al.,1994; Vanden Berghe et al., 1998). PCR fragments encoding TRADD and RIPwere cloned in pCDNA3 (Invitrogen, Carlsbad, Calif.) in frame with aC-terminal E-tag. Transfection and reporter assay was carried out asdescribed above.

[0115] NF-κB can be activated in 293 T cells by TNF treatment as well asby overexpression of specific proteins of the TNF-receptor complex,including TRADD, RIP, TRAF2 and NIK (Rothe et al.,1995; Malinin et al.,1997; Hsu et al., 1995; Ting et al., 1996). The latter associates withand activates IκB kinase complex which leads to IκB phosphorylation.This is a signal for ubiquitination and degradation of IκB, thusreleasing NF-κB which then translocates to the nucleus. Co-transfectionof expression plasmids encoding these TNF-receptor associated proteinstogether with the expression plasmids encoding full length ABIN, showedthat the latter completely inhibited NF-κB activation induced by TRADDor RIP, and partially inhibited TRAF2-induced NF-κB activation. Incontrast, no clear difference was observed when NF-κB-dependent reportergene expression was induced by NIK or more directly by overexpression ofthe p65 subunit of NF-κB (FIG. 8, FIG. 9). These results suggest thatABIN inhibits TNF-induced NF-κB activation at a level preceding theactivation of the NIK-IκB kinase steps, for example at the level ofTRAF2 in the TNF-receptor complex.

[0116] As members of the TRAF family mediate NF-κB activation by severalother stimuli, including IL-1, lymphotoxin β, CD30 and CD40 (Rothe etal., 1995; Cao et al., 1996; Nakano et al., 1996; Aizawa et al., 1997;Ishada et al., 1996), ABIN might have the potential to inhibit NF-κBactivation in response to a wide range of inducers. Therefore, drugsthat mimic the activity of ABIN are likely to have therapeutic value ininflammatory and neurodegenerative diseases as well as in cancer andAIDS.

Example 7

[0117] Cell Transfection, Co-immunoprecipitation and Western Blotting.

[0118] 2×10⁶ human embryonic kidney 293T cells were plated on 10 cmPetri dishes and transiently transfected by calcium phosphate-DNAcoprecipitation. 24 hours after transfection, cells were lysed in 500 lof lysis buffer (50 mM Hepes pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40 and5 mM EDTA). Lysates were incubated with 5 l of rabbit anti-GFP antibody(Clontech) and immunocomplexes were immobilized on protein A-Trisacryl(Pierce). The latter were washed twice with lysis buffer and twice withlysis buffer containing 1M NaCl. Coprecipitating proteins were separatedby SDS-PAGE and analyzed by Western blotting with mouse anti-E-tagantibody (Pharmacia).

Example 8

[0119] NF-κB Dependent Reporter Gene Assay.

[0120] NF-κB activity was determined by the NF-κB dependent expressionof a luciferase reporter gene. Therefore, 293T cells were plated in6-well plates at 4×10⁵ cells per well and transiently transfected by thecalcium phosphate-DNA coprecipitation method. Each transfectioncontained 800 ng of the specific expression plasmids, as well as 100 ngof pNFconluc plasmid and 100 ng pUT651 plasmid. 24 hours aftertransfection, these transfectants were trypsinized and seeded on a24-well plate. Another 24 hours later, cells were either stimulated with1000 IU/ml hTNF or 7000 IU/ml IL-1 or left untreated. After 6 hours ofstimulation, cells were lysed in 200 μl lysis buffer and analyzed forluciferase and β-galactosidase activity. Luciferase values (luc) arenormalized with (β-galactosidase values (gal) and plotted as luc/gal.

Example 9

[0121] Site Specific Mutagenesis.

[0122] Site specific mutagenesis on ABIN was performed by overlap PCRreaction using primers which contain the desired mutations. The primersused were the mutation primers5′-GAATACCAGGAGGCGCAGATCCAGCGGCTCAATAAAGCTTTGGAGGAGGC-3′ (SEQ ID NO:11),5′-GTTGCTGAAAGAGGACGTCAAAATCTTTGAAGAGG-3′ (SEQ ID NO:12),5′-GCAGGTAAAAATCTTTGAAGAGAATGCCCAGAGGGAACG-3′ (SEQ ID NO:13), and5′-GCAGGTAAAAATCTTTGAAGAGGACTTCCAGAGGGAAC GGAGTGATGCGCAACGCATGCCCG-3′(SEQ ID NO:14), a forward primer located at the start codon and tworeverse primers, one hybridizing in the 3′ UTR and one in the codingregion. The XhoI-BstEII fragment of wild type ABIN(54-647) in pCAGGS wasexchanged with the same fragment of the PCR amplified mutated ABINcDNA's.

Example 10

[0123] Binding of ABIN With A20 is Not Sufficient for its NF-κBInhibiting Potential.

[0124] A two hybrid assay with A20 revealed another novel A20-bindingprotein which was also able to inhibit NF-κB activation uponoverexpression. BLAST searches with this novel protein, named ABIN-2,revealed no homology with any known protein. However, by comparison ofthe protein sequence of ABIN-2 with ABIN, two boxes of 19 (AA 423-441)and 21 (AA 475-495) amino acids long with 68% and 67% homology wereidentified respectively. Therefore, the contribution of these regions tothe binding with A20 and to the NF-κB inhibiting effects of ABIN wereanalyzed by site specific mutagenesis of a number of conserved aminoacids. Co-immunoprecipitation analysis after transient overexpression ofGFP or GFP/A20 together with wild type ABIN or its site specific mutants(ABIN-MUT1, ABIN-MUT2, ABIN-MUT3 and ABIN-MUT4) (SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17 and SEQ ID NO:18 respectively) in 293T cells, showedthat all of these mutants can still bind A20 (FIG. 10). On the otherhand, point mutations in the second box (ABIN-MUT2, ABIN-MUT3 andABIN-MUT4) completely abolished the ability of ABIN to block NF-κBactivation upon stimulation of 293T cells with TNF, even when higheramounts of these expression plasmids were transfected. In contrast, themutation in the first box (ABIN-MUT1) only slightly diminished the NF-κBinhibiting effect of ABIN (FIG. 11). Furthermore, point mutations in thesecond conserved motif dominantly interfered with the NF-κB inhibitingeffect of wild type ABIN (FIG. 12). ABIN-MUT2 and ABIN-MUT3 exhibit amore potent function as dominant negative mutants of ABIN, compared toABIN-MUT4. In these assays, comparable expression levels of thedifferent mutants and of wild type ABIN were obtained as judged byWestern blot analysis using anti-E tag antibody. These results suggestthat the second conserved region is involved in the NF-κB inhibitingeffects of ABIN, and that binding of ABIN with A20 as such is notsufficient for inhibition of NF-κB activation.

REFERENCES

[0125] Aizawa S, et al. Tumor necrosis factor receptor-associated factor(TRAF) 5 and TRAF2 are involved in CD30-mediated NF-κB activation. JBiol Chem. 272, 2042-2045. (1997)

[0126] Barnes P J, et al. Anti-inflammatory actions of steroids:molecular mechanisms. Trends Pharmacol Sci. 14(12): 436-441 (1993).

[0127] Barnes P J and Karin M. Nuclear factor-kappaB: a pivotaltranscription factor in chronic inflammatory diseases. N Engl J Med.336(15): 1066-1071 (1997).

[0128] Behl C, et al. Mechanism of amyloid beta protein induced neuronalcell death: current concepts and future perspectives. J Neural TransmSuppl. 49: 125-134 (1997).

[0129] Brand K, et al. Activated transcription factor nuclearfactor-kappa B is present in the atherosclerotic lesion. J Clin Invest.97(7): 1715-1722 (1996).

[0130] Brand K, et al. Dysregulation of monocytic nuclear factor-kappa Bby oxidized low-density lipoprotein. Arterioscler Thromb Vasc Biol.17(10): 1901-1909 (1997).

[0131] Cao Z, et al. TRAF6 is a signal transducer for interleukin-l.Nature. 383, 443-446. (1996)

[0132] De Valck D, et al. A20, an inhibitor of cell death,self-associates by its zinc finger domain. FEBS Lett. 384, 61-64. (1996)

[0133] De Valck D, et al. A20 inhibits NF-κB activation independently ofbinding to 14-3-3 proteins. Biochem Biophys Res Commun. 238, 590-594.(1997)

[0134] DiDonato J A, et al. A cytokine-responsive IκB kinase thatactivates the transcription factor NF-κB. Nature. 388, 548-554. (1997)

[0135] Dixit V M, et al. Tumor necrosis factor-alpha induction of novelgene products in human endothelial cells including a macrophage-specificchemotoxin. J Biol Chem. 265, 2973-2978. (1990)

[0136] Harris M. From negative factor to a critical role in viruspathogenesis : the changing fortunes of Nef. J Gen Virol. 77, 2379-2392.(1996)

[0137] Hsu H, et al. The TNF receptor 1-associated protein TRADD signalscell death and NF-κB activation. Cell. 81, 495-504. (1995)

[0138] Ishida T, et al. Identification of TRAF6, a novel tumor necrosisfactor receptor-associated factor protein that mediates signaling froman amino-terminal domain of the CD40 cytoplasmic region. J Biol Chem.271, 28745-28748. (1996)

[0139] Jaattela M, et al. A20 zinc finger protein inhibits TNF and IL-1signaling. J Immunol. 156, 1166-1173. (1996)

[0140] Kaltschmidt C, et al. Transcription factor NF-kappa B isactivated in microglia during experimental autoimmune encephalomyelitis.J Neuroimmunol. 55(1): 99-106. (1994)

[0141] Kimura A, et al. Detailed analysis of the mouse H-2Kb promoter:enhancer-like sequences and their role in the regulation of class I geneexpression. Cell 44(2): 261-272. (1986)

[0142] Kopp E B and Ghosh S. Inhibition of NF-kappa B by sodiumsalicylate and aspirin. Science. 265(5174): 956-959. (1994)

[0143] Krikos A, et al. Transcriptional activation of the tumor necrosisfactor alpha-inducible zinc finger protein, A20, is mediated by κBelements. J Biol Chem. 267, 17971-17976. (1992)

[0144] Luque I, et al. Rel/NF-kappa B and I kappa B factors inoncogenesis. Semin Cancer Biol. 8(2): 103-111. (1997)

[0145] Malanin N L, et al. MAP3K-related kinase involved in NF-κBinduction by TNF, CD95 and IL-1. Nature. 385, 540-544 (1997)

[0146] Mosialos G. The role of Rel/NF-kappa B proteins in viraloncogenesis and the regulation of viral transcription. Semin CancerBiol. 8(2): 121-129. (1997)

[0147] Nagase, T et al. Prediction of the coding sequences ofunidentified human genes. III. The coding sequences of 40 new genes(KIAA0081-KIAA0120) deduced by analysis of cDNA clones from human cellline KG-1. DNA Res.2(1), 37-43 (1995).

[0148] Nakano H, et al. TRAF5, an activator of NF-κB and putative signaltransducer for the lymphotoxin-( receptor. J Biol Chem. 271,14661-14664. (1996)

[0149] Niwa H. et al. Efficient selection for high-expressiontransfectants with a novel eukaryotic vector. Gene. 108, 193-199. (1991)

[0150] Opipari, A. W. et al. The A20 cDNA induced by tumor necrosisfactor alpha encodes a novel type of zinc finger protein. J. Biol. Chem.265, 14705-14708, (1990).

[0151] Pahl H L, et al. The immunosuppressive fungal metabolitegliotoxin specifically inhibits transcription factor NF-kappaB. J ExpMed. 183(4): 1829-1840. (1996)

[0152] Regnier C H, et al. Identification and characterization of an IκBkinase. Cell. 90, 373-383. (1997) Remick D G. Applied molecular biologyof sepsis. J Crit Care. 10(4): 198-212. (1995)

[0153] Rothe M, et al. TRAF2-mediated activation of NF-κB by TNFreceptor 2 and CD40. Science. 269, 1424-1427. (1995)

[0154] Rothe M, et al. A novel family of putative signal transducersassociated with the cytoplasmic domain of the 75 kDa tumor necrosisfactor receptor. Cell. 78, 681-692. (1994)

[0155] Schreck R, et al. Reactive oxygen intermediates as apparentlywidely used messengers in the activation of the NF-kappa B transcriptionfactor and HIV-1. EMBO J. 10(8): 2247-2258 (1991)

[0156] Song H Y, et al. The tumor necrosis factor-inducible zinc fingerprotein A20 interacts with TRAF 1/TRAF2 and inhibits NF-κB activation.Proc Natl Acad Sci USA. 93, 6721-6725. (1996)

[0157] Tewari et al., Lymphoid expression and regulation of A20, aninhibitor of programmed cell death; J.Imnmunol. 1995, 154: 1699-1706.

[0158] Ting A T, et al. RIP mediates tumor necrosis factor receptor 1activation of NF-κB but not Fas/APO-1-initiated apoptosis. EMBO J. 15,6189-6196. (1996)

[0159] Vanden Berghe W. Et al. p38 and extracellular signal-regulatedkinase mitogen-activated protein kinase pathways are required fornuclear factor-kappaB p65 transactivation mediated by tumor necrosisfactor. J Biol Chem 273 (6): 3285-3290. (1998)

[0160] Wang P, et al. Interleukin (IL)-10 inhibits nuclear factor kappaB (NF kappa B) activation in human monocytes. Il-10 and IL-4 suppresscytokine synthesis by different mechanisms. J Biol Chem. 270(16):9558-9563. (1995)

[0161] Woronicz J D, et al. IκB kinase-beta : NF-κB activation andcomplex formation with IκB kinase-α and NIK. Science. 278,866-869.(1997)

[0162] Yeh W C. Et al. Early lethality, functional NF-kappaB activation,and increased sensitivity to TNF-induced cell death in TRAF2-deficientmice. Immunity 7(5): 715-725. (1997)

[0163] Zandi E, et al. The IκB kinase complex (IKK) contains two kinasesubunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κBactivation. Cell. 91, 243-252. (1997)

1 19 1 2812 DNA Mus musculus Intron (81)..(116) CDS (117)..(2060) 1cacagggagg catggccgca ctcactgggc acatcttcag atcacctcgt gcattctcgg 60atgagtgacc tgggctgaag ctaggcggcc gtcacggcag gggttgagcc accctc atg 119Met 1 gaa ggg aga gga ccc tac cgg atc tac gac cca ggg ggc agc acg cct167 Glu Gly Arg Gly Pro Tyr Arg Ile Tyr Asp Pro Gly Gly Ser Thr Pro 5 1015 ctg gga gag gtg tcc gca gct ttt gaa cgt cta gtg gag gag aat act 215Leu Gly Glu Val Ser Ala Ala Phe Glu Arg Leu Val Glu Glu Asn Thr 20 25 30cgg ctg aag gga aaa atg caa ggg ata aag atg tta ggg gag ctt ctg 263 ArgLeu Lys Gly Lys Met Gln Gly Ile Lys Met Leu Gly Glu Leu Leu 35 40 45 gaggag tct cag atg gaa gcg tcc aga ctc cgg cag aag gca gag gag 311 Glu GluSer Gln Met Glu Ala Ser Arg Leu Arg Gln Lys Ala Glu Glu 50 55 60 65 ctggtc aag gac agc gag ctg tca cca ccg aca tct gcc ccc tcc ttg 359 Leu ValLys Asp Ser Glu Leu Ser Pro Pro Thr Ser Ala Pro Ser Leu 70 75 80 gtc tccttt gat gac ctg gct gag ctc aca gga cag gat aca aag gtc 407 Val Ser PheAsp Asp Leu Ala Glu Leu Thr Gly Gln Asp Thr Lys Val 85 90 95 cag gta catcct gct acc agc act gcc gcc acc acc acc gcc acc gcc 455 Gln Val His ProAla Thr Ser Thr Ala Ala Thr Thr Thr Ala Thr Ala 100 105 110 acc acg ggaaac tcc atg gag aag ccc gag cca gcc tcc aaa tct ccg 503 Thr Thr Gly AsnSer Met Glu Lys Pro Glu Pro Ala Ser Lys Ser Pro 115 120 125 tcc aat ggcgcc tcc tcg gac ttt gaa gtg gtc cct act gag gag cag 551 Ser Asn Gly AlaSer Ser Asp Phe Glu Val Val Pro Thr Glu Glu Gln 130 135 140 145 aat tcaccc gaa act ggc agc cac cct acg aac atg atg gac ctg ggg 599 Asn Ser ProGlu Thr Gly Ser His Pro Thr Asn Met Met Asp Leu Gly 150 155 160 ccc ccaccc cca gag gac agc aac ctg aag ctc cac ctg cag cgc ctg 647 Pro Pro ProPro Glu Asp Ser Asn Leu Lys Leu His Leu Gln Arg Leu 165 170 175 gag accacc ctt agc gtg tgt gca gag gag cca gac cac agc cag ctc 695 Glu Thr ThrLeu Ser Val Cys Ala Glu Glu Pro Asp His Ser Gln Leu 180 185 190 ttc acccac ctg ggc cgc atg gcc ctc gag ttc aac agg ttg gcc tcc 743 Phe Thr HisLeu Gly Arg Met Ala Leu Glu Phe Asn Arg Leu Ala Ser 195 200 205 aaa gtgcat aaa aat gag cag cgc acc tcc atc ctg cag acc tta tgt 791 Lys Val HisLys Asn Glu Gln Arg Thr Ser Ile Leu Gln Thr Leu Cys 210 215 220 225 gagcag ctg cgc cag gag aat gaa gcc ctg aag gcc aag ctg gac aag 839 Glu GlnLeu Arg Gln Glu Asn Glu Ala Leu Lys Ala Lys Leu Asp Lys 230 235 240 ggcctg gaa cag cgg gat ctg gct gct gag agg ctg cgg gag gaa aac 887 Gly LeuGlu Gln Arg Asp Leu Ala Ala Glu Arg Leu Arg Glu Glu Asn 245 250 255 acggag ctc aag aaa ctg ttg atg aac agc agc tgc aaa gag gga ctc 935 Thr GluLeu Lys Lys Leu Leu Met Asn Ser Ser Cys Lys Glu Gly Leu 260 265 270 tgtggg cag ccc agc tcc cca aag cca gag ggt gct ggc aag aag ggc 983 Cys GlyGln Pro Ser Ser Pro Lys Pro Glu Gly Ala Gly Lys Lys Gly 275 280 285 gtggct gga cag cag cag gcc agt gtg atg gcg agt aaa gtc cct gaa 1031 Val AlaGly Gln Gln Gln Ala Ser Val Met Ala Ser Lys Val Pro Glu 290 295 300 305gcg ggg gcc ttt gga gca gct gag aag aaa gtg aag ttg cta gaa cag 1079 AlaGly Ala Phe Gly Ala Ala Glu Lys Lys Val Lys Leu Leu Glu Gln 310 315 320caa cgc atg gag ctg ctg gaa gtg aac aag cag tgg gac cag cat ttc 1127 GlnArg Met Glu Leu Leu Glu Val Asn Lys Gln Trp Asp Gln His Phe 325 330 335cgg tcc atg aag cag cag tat gag cag aag atc aca gag ctt cgc cag 1175 ArgSer Met Lys Gln Gln Tyr Glu Gln Lys Ile Thr Glu Leu Arg Gln 340 345 350aag ctg gtg gac ctg cag aaa cag gta act gag ctg gag gcc gaa cgg 1223 LysLeu Val Asp Leu Gln Lys Gln Val Thr Glu Leu Glu Ala Glu Arg 355 360 365gag cag aag cag cgt gac ttt gac cgg aaa ctc ctc ctg gcc aaa tcg 1271 GluGln Lys Gln Arg Asp Phe Asp Arg Lys Leu Leu Leu Ala Lys Ser 370 375 380385 aag ata gag atg gaa gag acc gac aag gag cag ctg aca gca gag gcc 1319Lys Ile Glu Met Glu Glu Thr Asp Lys Glu Gln Leu Thr Ala Glu Ala 390 395400 aag gaa ctg cgc cag aag gtc agg tac cta cag gat cag ctg agc ccg 1367Lys Glu Leu Arg Gln Lys Val Arg Tyr Leu Gln Asp Gln Leu Ser Pro 405 410415 ctc aca agg caa cga gaa tac cag gag aag gag atc cag cgg ctc aat 1415Leu Thr Arg Gln Arg Glu Tyr Gln Glu Lys Glu Ile Gln Arg Leu Asn 420 425430 aag gcc ctg gag gag gcc ctc agc atc cag gcc tct cca tca tct ccg 1463Lys Ala Leu Glu Glu Ala Leu Ser Ile Gln Ala Ser Pro Ser Ser Pro 435 440445 cct gca gct ttt ggg agt cca gaa ggc gtt ggg ggc cat ctg agg aag 1511Pro Ala Ala Phe Gly Ser Pro Glu Gly Val Gly Gly His Leu Arg Lys 450 455460 465 cag gaa cta gtg aca cag aat gag ttg ctg aaa cag cag gta aag atc1559 Gln Glu Leu Val Thr Gln Asn Glu Leu Leu Lys Gln Gln Val Lys Ile 470475 480 ttt gaa gag gac ttc cag agg gaa cgg agt gac cgt gaa cgc atg aat1607 Phe Glu Glu Asp Phe Gln Arg Glu Arg Ser Asp Arg Glu Arg Met Asn 485490 495 gaa gag aag gag gag ctg aag aag caa gta gag aag ctg cag gcc cag1655 Glu Glu Lys Glu Glu Leu Lys Lys Gln Val Glu Lys Leu Gln Ala Gln 500505 510 gtc acc ctg act aat gcc cag ctc aaa act ctc aaa gag gag gag aag1703 Val Thr Leu Thr Asn Ala Gln Leu Lys Thr Leu Lys Glu Glu Glu Lys 515520 525 gcc aag gaa gcc ctc aaa cag cag aag agg aaa gca aag gct tcg gga1751 Ala Lys Glu Ala Leu Lys Gln Gln Lys Arg Lys Ala Lys Ala Ser Gly 530535 540 545 gag cgc tac cac atg gaa ccc cac cct gag cac gtc tgc ggc gcctat 1799 Glu Arg Tyr His Met Glu Pro His Pro Glu His Val Cys Gly Ala Tyr550 555 560 ccc tat gcc tac cca ccc atg cca gcc atg gta cct cac cat gcctac 1847 Pro Tyr Ala Tyr Pro Pro Met Pro Ala Met Val Pro His His Ala Tyr565 570 575 aag gac tgg tcc cag atc cga tac cct cca ccc cct gtg ccc atggag 1895 Lys Asp Trp Ser Gln Ile Arg Tyr Pro Pro Pro Pro Val Pro Met Glu580 585 590 cac ccg ccc cca cac ccc aac tct cgc ctc ttc cat ctg ccg gagtac 1943 His Pro Pro Pro His Pro Asn Ser Arg Leu Phe His Leu Pro Glu Tyr595 600 605 acc tgg cgt cca ccc tgt gca ggg att cgg aat cag agc tct caagtg 1991 Thr Trp Arg Pro Pro Cys Ala Gly Ile Arg Asn Gln Ser Ser Gln Val610 615 620 625 atg gac ccg ccc cca gac agg cct gca gag cca gag tct gcagac aat 2039 Met Asp Pro Pro Pro Asp Arg Pro Ala Glu Pro Glu Ser Ala AspAsn 630 635 640 gac tgt gat ggg ccc cag tga ggctgcagtg ggtcatttggttccaccttc 2090 Asp Cys Asp Gly Pro Gln 645 atctttcaga gccagctgacctcagattgc caaaagtttg aaggccatgt gcatgttctg 2150 tgtgacccaa gccttggcagaggagaggct gggatgggta gctggctcac atccccagcc 2210 aagcctcgaa ctgttgacaagaccagggag aatccaccca tgggcgccca ccaggttctt 2270 atggatgcaa gcaggagaagctcaacaccc tgcctcttgc caagacaagg aagcctcacc 2330 tggctttgac ctgccatccgttgctgaggc cactggcttc catcctaaga atgaggtgca 2390 acaagacccc attctcacagaacctcaaag acttggttcc aggctctcca gagaccatac 2450 ccaactcatg tgcatgtgccgtttttgctt caagctcagt agcaggacct gccccgagcc 2510 ccctgctcct tgcccctctgtgaggagtta cggagagggc tttgtctcta gagcagaaga 2570 gaatgatggg acggcctgatgctgtcatgc tctccactgc acctgtggca gcctcctgag 2630 agccaccaag atctgggatgaaggccacac cagccatgtc tgctgaaggg ccccagactg 2690 agatgactcc ggcctccacagttagatgtt tatggtgcca gaggtctata ttaaggtagc 2750 tgtctgttgc taggcagccgtttgcacaaa tcttggacat aaatccaact tgaagatcaa 2810 aa 2812 2 647 PRT Musmusculus 2 Met Glu Gly Arg Gly Pro Tyr Arg Ile Tyr Asp Pro Gly Gly SerThr 1 5 10 15 Pro Leu Gly Glu Val Ser Ala Ala Phe Glu Arg Leu Val GluGlu Asn 20 25 30 Thr Arg Leu Lys Gly Lys Met Gln Gly Ile Lys Met Leu GlyGlu Leu 35 40 45 Leu Glu Glu Ser Gln Met Glu Ala Ser Arg Leu Arg Gln LysAla Glu 50 55 60 Glu Leu Val Lys Asp Ser Glu Leu Ser Pro Pro Thr Ser AlaPro Ser 65 70 75 80 Leu Val Ser Phe Asp Asp Leu Ala Glu Leu Thr Gly GlnAsp Thr Lys 85 90 95 Val Gln Val His Pro Ala Thr Ser Thr Ala Ala Thr ThrThr Ala Thr 100 105 110 Ala Thr Thr Gly Asn Ser Met Glu Lys Pro Glu ProAla Ser Lys Ser 115 120 125 Pro Ser Asn Gly Ala Ser Ser Asp Phe Glu ValVal Pro Thr Glu Glu 130 135 140 Gln Asn Ser Pro Glu Thr Gly Ser His ProThr Asn Met Met Asp Leu 145 150 155 160 Gly Pro Pro Pro Pro Glu Asp SerAsn Leu Lys Leu His Leu Gln Arg 165 170 175 Leu Glu Thr Thr Leu Ser ValCys Ala Glu Glu Pro Asp His Ser Gln 180 185 190 Leu Phe Thr His Leu GlyArg Met Ala Leu Glu Phe Asn Arg Leu Ala 195 200 205 Ser Lys Val His LysAsn Glu Gln Arg Thr Ser Ile Leu Gln Thr Leu 210 215 220 Cys Glu Gln LeuArg Gln Glu Asn Glu Ala Leu Lys Ala Lys Leu Asp 225 230 235 240 Lys GlyLeu Glu Gln Arg Asp Leu Ala Ala Glu Arg Leu Arg Glu Glu 245 250 255 AsnThr Glu Leu Lys Lys Leu Leu Met Asn Ser Ser Cys Lys Glu Gly 260 265 270Leu Cys Gly Gln Pro Ser Ser Pro Lys Pro Glu Gly Ala Gly Lys Lys 275 280285 Gly Val Ala Gly Gln Gln Gln Ala Ser Val Met Ala Ser Lys Val Pro 290295 300 Glu Ala Gly Ala Phe Gly Ala Ala Glu Lys Lys Val Lys Leu Leu Glu305 310 315 320 Gln Gln Arg Met Glu Leu Leu Glu Val Asn Lys Gln Trp AspGln His 325 330 335 Phe Arg Ser Met Lys Gln Gln Tyr Glu Gln Lys Ile ThrGlu Leu Arg 340 345 350 Gln Lys Leu Val Asp Leu Gln Lys Gln Val Thr GluLeu Glu Ala Glu 355 360 365 Arg Glu Gln Lys Gln Arg Asp Phe Asp Arg LysLeu Leu Leu Ala Lys 370 375 380 Ser Lys Ile Glu Met Glu Glu Thr Asp LysGlu Gln Leu Thr Ala Glu 385 390 395 400 Ala Lys Glu Leu Arg Gln Lys ValArg Tyr Leu Gln Asp Gln Leu Ser 405 410 415 Pro Leu Thr Arg Gln Arg GluTyr Gln Glu Lys Glu Ile Gln Arg Leu 420 425 430 Asn Lys Ala Leu Glu GluAla Leu Ser Ile Gln Ala Ser Pro Ser Ser 435 440 445 Pro Pro Ala Ala PheGly Ser Pro Glu Gly Val Gly Gly His Leu Arg 450 455 460 Lys Gln Glu LeuVal Thr Gln Asn Glu Leu Leu Lys Gln Gln Val Lys 465 470 475 480 Ile PheGlu Glu Asp Phe Gln Arg Glu Arg Ser Asp Arg Glu Arg Met 485 490 495 AsnGlu Glu Lys Glu Glu Leu Lys Lys Gln Val Glu Lys Leu Gln Ala 500 505 510Gln Val Thr Leu Thr Asn Ala Gln Leu Lys Thr Leu Lys Glu Glu Glu 515 520525 Lys Ala Lys Glu Ala Leu Lys Gln Gln Lys Arg Lys Ala Lys Ala Ser 530535 540 Gly Glu Arg Tyr His Met Glu Pro His Pro Glu His Val Cys Gly Ala545 550 555 560 Tyr Pro Tyr Ala Tyr Pro Pro Met Pro Ala Met Val Pro HisHis Ala 565 570 575 Tyr Lys Asp Trp Ser Gln Ile Arg Tyr Pro Pro Pro ProVal Pro Met 580 585 590 Glu His Pro Pro Pro His Pro Asn Ser Arg Leu PheHis Leu Pro Glu 595 600 605 Tyr Thr Trp Arg Pro Pro Cys Ala Gly Ile ArgAsn Gln Ser Ser Gln 610 615 620 Val Met Asp Pro Pro Pro Asp Arg Pro AlaGlu Pro Glu Ser Ala Asp 625 630 635 640 Asn Asp Cys Asp Gly Pro Gln 6453 627 PRT Mus musculus 3 Val Ser Ala Ala Phe Glu Arg Leu Val Glu Glu AsnThr Arg Leu Lys 1 5 10 15 Gly Lys Met Gln Gly Ile Lys Met Leu Gly GluLeu Leu Glu Glu Ser 20 25 30 Gln Met Glu Ala Ser Arg Leu Arg Gln Lys AlaGlu Glu Leu Val Lys 35 40 45 Asp Ser Glu Leu Ser Pro Pro Thr Ser Ala ProSer Leu Val Ser Phe 50 55 60 Asp Asp Leu Ala Glu Leu Thr Gly Gln Asp ThrLys Val Gln Val His 65 70 75 80 Pro Ala Thr Ser Thr Ala Ala Thr Thr ThrAla Thr Ala Thr Thr Gly 85 90 95 Asn Ser Met Glu Lys Pro Glu Pro Ala SerLys Ser Pro Ser Asn Gly 100 105 110 Ala Ser Ser Asp Phe Glu Val Val ProThr Glu Glu Gln Asn Ser Pro 115 120 125 Glu Thr Gly Ser His Pro Thr AsnMet Met Asp Leu Gly Pro Pro Pro 130 135 140 Pro Glu Asp Ser Asn Leu LysLeu His Leu Gln Arg Leu Glu Thr Thr 145 150 155 160 Leu Ser Val Cys AlaGlu Glu Pro Asp His Ser Gln Leu Phe Thr His 165 170 175 Leu Gly Arg MetAla Leu Glu Phe Asn Arg Leu Ala Ser Lys Val His 180 185 190 Lys Asn GluGln Arg Thr Ser Ile Leu Gln Thr Leu Cys Glu Gln Leu 195 200 205 Arg GlnGlu Asn Glu Ala Leu Lys Ala Lys Leu Asp Lys Gly Leu Glu 210 215 220 GlnArg Asp Leu Ala Ala Glu Arg Leu Arg Glu Glu Asn Thr Glu Leu 225 230 235240 Lys Lys Leu Leu Met Asn Ser Ser Cys Lys Glu Gly Leu Cys Gly Gln 245250 255 Pro Ser Ser Pro Lys Pro Glu Gly Ala Gly Lys Lys Gly Val Ala Gly260 265 270 Gln Gln Gln Ala Ser Val Met Ala Ser Lys Val Pro Glu Ala GlyAla 275 280 285 Phe Gly Ala Ala Glu Lys Lys Val Lys Leu Leu Glu Gln GlnArg Met 290 295 300 Glu Leu Leu Glu Val Asn Lys Gln Trp Asp Gln His PheArg Ser Met 305 310 315 320 Lys Gln Gln Tyr Glu Gln Lys Ile Thr Glu LeuArg Gln Lys Leu Val 325 330 335 Asp Leu Gln Lys Gln Val Thr Glu Leu GluAla Glu Arg Glu Gln Lys 340 345 350 Gln Arg Asp Phe Asp Arg Lys Leu LeuLeu Ala Lys Ser Lys Ile Glu 355 360 365 Met Glu Glu Thr Asp Lys Glu GlnLeu Thr Ala Glu Ala Lys Glu Leu 370 375 380 Arg Gln Lys Val Arg Tyr LeuGln Asp Gln Leu Ser Pro Leu Thr Arg 385 390 395 400 Gln Arg Glu Tyr GlnGlu Lys Glu Ile Gln Arg Leu Asn Lys Ala Leu 405 410 415 Glu Glu Ala LeuSer Ile Gln Ala Ser Pro Ser Ser Pro Pro Ala Ala 420 425 430 Phe Gly SerPro Glu Gly Val Gly Gly His Leu Arg Lys Gln Glu Leu 435 440 445 Val ThrGln Asn Glu Leu Leu Lys Gln Gln Val Lys Ile Phe Glu Glu 450 455 460 AspPhe Gln Arg Glu Arg Ser Asp Arg Glu Arg Met Asn Glu Glu Lys 465 470 475480 Glu Glu Leu Lys Lys Gln Val Glu Lys Leu Gln Ala Gln Val Thr Leu 485490 495 Thr Asn Ala Gln Leu Lys Thr Leu Lys Glu Glu Glu Lys Ala Lys Glu500 505 510 Ala Leu Lys Gln Gln Lys Arg Lys Ala Lys Ala Ser Gly Glu ArgTyr 515 520 525 His Met Glu Pro His Pro Glu His Val Cys Gly Ala Tyr ProTyr Ala 530 535 540 Tyr Pro Pro Met Pro Ala Met Val Pro His His Ala TyrLys Asp Trp 545 550 555 560 Ser Gln Ile Arg Tyr Pro Pro Pro Pro Val ProMet Glu His Pro Pro 565 570 575 Pro His Pro Asn Ser Arg Leu Phe His LeuPro Glu Tyr Thr Trp Arg 580 585 590 Pro Pro Cys Ala Gly Ile Arg Asn GlnSer Ser Gln Val Met Asp Pro 595 600 605 Pro Pro Asp Arg Pro Ala Glu ProGlu Ser Ala Asp Asn Asp Cys Asp 610 615 620 Gly Pro Gln 625 4 574 PRTMus musculus 4 Pro Pro Thr Ser Ala Pro Ser Leu Val Ser Phe Asp Asp LeuAla Glu 1 5 10 15 Leu Thr Gly Gln Asp Thr Lys Val Gln Val His Pro AlaThr Ser Thr 20 25 30 Ala Ala Thr Thr Thr Ala Thr Ala Thr Thr Gly Asn SerMet Glu Lys 35 40 45 Pro Glu Pro Ala Ser Lys Ser Pro Ser Asn Gly Ala SerSer Asp Phe 50 55 60 Glu Val Val Pro Thr Glu Glu Gln Asn Ser Pro Glu ThrGly Ser His 65 70 75 80 Pro Thr Asn Met Met Asp Leu Gly Pro Pro Pro ProGlu Asp Ser Asn 85 90 95 Leu Lys Leu His Leu Gln Arg Leu Glu Thr Thr LeuSer Val Cys Ala 100 105 110 Glu Glu Pro Asp His Ser Gln Leu Phe Thr HisLeu Gly Arg Met Ala 115 120 125 Leu Glu Phe Asn Arg Leu Ala Ser Lys ValHis Lys Asn Glu Gln Arg 130 135 140 Thr Ser Ile Leu Gln Thr Leu Cys GluGln Leu Arg Gln Glu Asn Glu 145 150 155 160 Ala Leu Lys Ala Lys Leu AspLys Gly Leu Glu Gln Arg Asp Leu Ala 165 170 175 Ala Glu Arg Leu Arg GluGlu Asn Thr Glu Leu Lys Lys Leu Leu Met 180 185 190 Asn Ser Ser Cys LysGlu Gly Leu Cys Gly Gln Pro Ser Ser Pro Lys 195 200 205 Pro Glu Gly AlaGly Lys Lys Gly Val Ala Gly Gln Gln Gln Ala Ser 210 215 220 Val Met AlaSer Lys Val Pro Glu Ala Gly Ala Phe Gly Ala Ala Glu 225 230 235 240 LysLys Val Lys Leu Leu Glu Gln Gln Arg Met Glu Leu Leu Glu Val 245 250 255Asn Lys Gln Trp Asp Gln His Phe Arg Ser Met Lys Gln Gln Tyr Glu 260 265270 Gln Lys Ile Thr Glu Leu Arg Gln Lys Leu Val Asp Leu Gln Lys Gln 275280 285 Val Thr Glu Leu Glu Ala Glu Arg Glu Gln Lys Gln Arg Asp Phe Asp290 295 300 Arg Lys Leu Leu Leu Ala Lys Ser Lys Ile Glu Met Glu Glu ThrAsp 305 310 315 320 Lys Glu Gln Leu Thr Ala Glu Ala Lys Glu Leu Arg GlnLys Val Arg 325 330 335 Tyr Leu Gln Asp Gln Leu Ser Pro Leu Thr Arg GlnArg Glu Tyr Gln 340 345 350 Glu Lys Glu Ile Gln Arg Leu Asn Lys Ala LeuGlu Glu Ala Leu Ser 355 360 365 Ile Gln Ala Ser Pro Ser Ser Pro Pro AlaAla Phe Gly Ser Pro Glu 370 375 380 Gly Val Gly Gly His Leu Arg Lys GlnGlu Leu Val Thr Gln Asn Glu 385 390 395 400 Leu Leu Lys Gln Gln Val LysIle Phe Glu Glu Asp Phe Gln Arg Glu 405 410 415 Arg Ser Asp Arg Glu ArgMet Asn Glu Glu Lys Glu Glu Leu Lys Lys 420 425 430 Gln Val Glu Lys LeuGln Ala Gln Val Thr Leu Thr Asn Ala Gln Leu 435 440 445 Lys Thr Leu LysGlu Glu Glu Lys Ala Lys Glu Ala Leu Lys Gln Gln 450 455 460 Lys Arg LysAla Lys Ala Ser Gly Glu Arg Tyr His Met Glu Pro His 465 470 475 480 ProGlu His Val Cys Gly Ala Tyr Pro Tyr Ala Tyr Pro Pro Met Pro 485 490 495Ala Met Val Pro His His Ala Tyr Lys Asp Trp Ser Gln Ile Arg Tyr 500 505510 Pro Pro Pro Pro Val Pro Met Glu His Pro Pro Pro His Pro Asn Ser 515520 525 Arg Leu Phe His Leu Pro Glu Tyr Thr Trp Arg Pro Pro Cys Ala Gly530 535 540 Ile Arg Asn Gln Ser Ser Gln Val Met Asp Pro Pro Pro Asp ArgPro 545 550 555 560 Ala Glu Pro Glu Ser Ala Asp Asn Asp Cys Asp Gly ProGln 565 570 5 1967 DNA Mus musculus CDS (83)..(1375) 5 aaactttccgggaaggctgg ttttcgctcc ccctgtgtgg agaagttgga gacgcccaag 60 tccccacggaaggcctacag cc atg tcg tct ggg gac cca agg tct ggt aga 112 Met Ser SerGly Asp Pro Arg Ser Gly Arg 1 5 10 cag gac ggg gcc ccg cgt gcg gcc gcagcg ctc tgt ggc ctg tac cac 160 Gln Asp Gly Ala Pro Arg Ala Ala Ala AlaLeu Cys Gly Leu Tyr His 15 20 25 gag gcc ggc cag caa cta cag cgc ctg aaggat cag ctg gcc gcg cgt 208 Glu Ala Gly Gln Gln Leu Gln Arg Leu Lys AspGln Leu Ala Ala Arg 30 35 40 gac gcc ctc atc gcg agc ctc cgc acc cgc ctcgcg gct ctg gaa ggg 256 Asp Ala Leu Ile Ala Ser Leu Arg Thr Arg Leu AlaAla Leu Glu Gly 45 50 55 cac acg gcg ccg tca ctc gtg gac gca ctt ctg gatcag gtg gag cgc 304 His Thr Ala Pro Ser Leu Val Asp Ala Leu Leu Asp GlnVal Glu Arg 60 65 70 ttc cgt gag cag ctg cga cga cag gag gaa ggc gct tcggag acc cag 352 Phe Arg Glu Gln Leu Arg Arg Gln Glu Glu Gly Ala Ser GluThr Gln 75 80 85 90 ctg cgg cag gaa gtt gaa aga ctt acg gag cgt cta gaggaa aaa gag 400 Leu Arg Gln Glu Val Glu Arg Leu Thr Glu Arg Leu Glu GluLys Glu 95 100 105 agg gag atg caa cag ctg atg agc cag cct cag cat gagcaa gag aag 448 Arg Glu Met Gln Gln Leu Met Ser Gln Pro Gln His Glu GlnGlu Lys 110 115 120 gag gta gtc ttg ctt cgg cga agt gtg gca gag aag gagaaa gcc agg 496 Glu Val Val Leu Leu Arg Arg Ser Val Ala Glu Lys Glu LysAla Arg 125 130 135 gcc gcc agt gat gtt ctg tgc cgc tcc ttg gct gat gagacc cac caa 544 Ala Ala Ser Asp Val Leu Cys Arg Ser Leu Ala Asp Glu ThrHis Gln 140 145 150 ctg cgc agg aca ttg gca gcc act gcc cac atg tgc caacat ctg gcc 592 Leu Arg Arg Thr Leu Ala Ala Thr Ala His Met Cys Gln HisLeu Ala 155 160 165 170 aaa tgt ctg gat gaa cga cag tgt gca cag gga gacgct ggg gag aaa 640 Lys Cys Leu Asp Glu Arg Gln Cys Ala Gln Gly Asp AlaGly Glu Lys 175 180 185 agc cct gct gag cta gag caa aca agc agc gat gcttct ggc cag agt 688 Ser Pro Ala Glu Leu Glu Gln Thr Ser Ser Asp Ala SerGly Gln Ser 190 195 200 gtt att aag aag tta cag gaa gaa aat cga ctg ttaaaa cag aag gtg 736 Val Ile Lys Lys Leu Gln Glu Glu Asn Arg Leu Leu LysGln Lys Val 205 210 215 act cat gta gaa gac ctc aat gct aag tgg cag cgttat gat gca agt 784 Thr His Val Glu Asp Leu Asn Ala Lys Trp Gln Arg TyrAsp Ala Ser 220 225 230 agg gac gaa tat gtg aag ggg ttg cat gcc cag ctaaag agg cgg cag 832 Arg Asp Glu Tyr Val Lys Gly Leu His Ala Gln Leu LysArg Arg Gln 235 240 245 250 gtc cct ctg gag cct gag ctg atg aag aag gagatt tcc cga ctt aac 880 Val Pro Leu Glu Pro Glu Leu Met Lys Lys Glu IleSer Arg Leu Asn 255 260 265 aga cag ttg gag gag aaa ata agt gac tgt gcggaa gca aac cag gag 928 Arg Gln Leu Glu Glu Lys Ile Ser Asp Cys Ala GluAla Asn Gln Glu 270 275 280 ctg aca gcc atg agg atg tcc cgg gac act gcgctg gag cga gtg cag 976 Leu Thr Ala Met Arg Met Ser Arg Asp Thr Ala LeuGlu Arg Val Gln 285 290 295 atg cta gaa cag cag att ctt gct tac aag gatgac ttc aaa tca gaa 1024 Met Leu Glu Gln Gln Ile Leu Ala Tyr Lys Asp AspPhe Lys Ser Glu 300 305 310 agg gca gat cgg gaa cga gcg cac agt agg attcaa gag ctg gag gaa 1072 Arg Ala Asp Arg Glu Arg Ala His Ser Arg Ile GlnGlu Leu Glu Glu 315 320 325 330 aag atc atg tcc ttg atg tac caa gtg tcccag aga cag gac tcc cgg 1120 Lys Ile Met Ser Leu Met Tyr Gln Val Ser GlnArg Gln Asp Ser Arg 335 340 345 gag cca gga ccc tgt cgg att cat acg gggaac aaa act gcc aag tac 1168 Glu Pro Gly Pro Cys Arg Ile His Thr Gly AsnLys Thr Ala Lys Tyr 350 355 360 tta gag atg gat gca ctg gag cat gtg acccct ggc ggc tgg agg cct 1216 Leu Glu Met Asp Ala Leu Glu His Val Thr ProGly Gly Trp Arg Pro 365 370 375 gag tct agg tcc caa cag atg gaa cct tctgca gag ggt ggg cat gtg 1264 Glu Ser Arg Ser Gln Gln Met Glu Pro Ser AlaGlu Gly Gly His Val 380 385 390 tgc aca gcc cag aga ggt cag ggt gac cttcag tgc cct cat tgc ctg 1312 Cys Thr Ala Gln Arg Gly Gln Gly Asp Leu GlnCys Pro His Cys Leu 395 400 405 410 cgg tgc ttc agt gat gag caa ggc gaggca ttc ctc agg cac ctg tct 1360 Arg Cys Phe Ser Asp Glu Gln Gly Glu AlaPhe Leu Arg His Leu Ser 415 420 425 gag tgc tgc caa tga gccagacattgcccgtgtga cccatgacca ccatagctgc 1415 Glu Cys Cys Gln 430 tctaagggactgggaggggt cctcagactc agttttcaac tcagtgtgtt gcattctcct 1475 gggatctagggcccaaatgg gcagggtcac tggaaggtca tcttgttttc atttgaccat 1535 ggtgagacttggtcagaggg aactattgac agagcaggag gaagagggtg gggtcaggga 1595 catcaagtggacatcagttt tgtctcacgt agagtttgga gtgagctgtc aattcaaagc 1655 tgcaagctatcagttgtggg aatattctga agcctgcttg cacctagagt tatgccactt 1715 gctggaaggggaagttgctg tgggagcagt gtgtcctctt tctagggtgg tagctccatc 1775 ctgttgagtagtgagataca ctccctgact ggtctgtgct gcattacagt tacatgatac 1835 actagaaccttcccaaactc agcagagcca cacagctgca tccagtacca tcaccctgca 1895 aaacacttgtatttccaaaa gggaaagcac ctttatttcc taatcattta tttttataat 1955 aaatggctttac 1967 6 430 PRT Mus musculus 6 Met Ser Ser Gly Asp Pro Arg Ser Gly ArgGln Asp Gly Ala Pro Arg 1 5 10 15 Ala Ala Ala Ala Leu Cys Gly Leu TyrHis Glu Ala Gly Gln Gln Leu 20 25 30 Gln Arg Leu Lys Asp Gln Leu Ala AlaArg Asp Ala Leu Ile Ala Ser 35 40 45 Leu Arg Thr Arg Leu Ala Ala Leu GluGly His Thr Ala Pro Ser Leu 50 55 60 Val Asp Ala Leu Leu Asp Gln Val GluArg Phe Arg Glu Gln Leu Arg 65 70 75 80 Arg Gln Glu Glu Gly Ala Ser GluThr Gln Leu Arg Gln Glu Val Glu 85 90 95 Arg Leu Thr Glu Arg Leu Glu GluLys Glu Arg Glu Met Gln Gln Leu 100 105 110 Met Ser Gln Pro Gln His GluGln Glu Lys Glu Val Val Leu Leu Arg 115 120 125 Arg Ser Val Ala Glu LysGlu Lys Ala Arg Ala Ala Ser Asp Val Leu 130 135 140 Cys Arg Ser Leu AlaAsp Glu Thr His Gln Leu Arg Arg Thr Leu Ala 145 150 155 160 Ala Thr AlaHis Met Cys Gln His Leu Ala Lys Cys Leu Asp Glu Arg 165 170 175 Gln CysAla Gln Gly Asp Ala Gly Glu Lys Ser Pro Ala Glu Leu Glu 180 185 190 GlnThr Ser Ser Asp Ala Ser Gly Gln Ser Val Ile Lys Lys Leu Gln 195 200 205Glu Glu Asn Arg Leu Leu Lys Gln Lys Val Thr His Val Glu Asp Leu 210 215220 Asn Ala Lys Trp Gln Arg Tyr Asp Ala Ser Arg Asp Glu Tyr Val Lys 225230 235 240 Gly Leu His Ala Gln Leu Lys Arg Arg Gln Val Pro Leu Glu ProGlu 245 250 255 Leu Met Lys Lys Glu Ile Ser Arg Leu Asn Arg Gln Leu GluGlu Lys 260 265 270 Ile Ser Asp Cys Ala Glu Ala Asn Gln Glu Leu Thr AlaMet Arg Met 275 280 285 Ser Arg Asp Thr Ala Leu Glu Arg Val Gln Met LeuGlu Gln Gln Ile 290 295 300 Leu Ala Tyr Lys Asp Asp Phe Lys Ser Glu ArgAla Asp Arg Glu Arg 305 310 315 320 Ala His Ser Arg Ile Gln Glu Leu GluGlu Lys Ile Met Ser Leu Met 325 330 335 Tyr Gln Val Ser Gln Arg Gln AspSer Arg Glu Pro Gly Pro Cys Arg 340 345 350 Ile His Thr Gly Asn Lys ThrAla Lys Tyr Leu Glu Met Asp Ala Leu 355 360 365 Glu His Val Thr Pro GlyGly Trp Arg Pro Glu Ser Arg Ser Gln Gln 370 375 380 Met Glu Pro Ser AlaGlu Gly Gly His Val Cys Thr Ala Gln Arg Gly 385 390 395 400 Gln Gly AspLeu Gln Cys Pro His Cys Leu Arg Cys Phe Ser Asp Glu 405 410 415 Gln GlyGlu Ala Phe Leu Arg His Leu Ser Glu Cys Cys Gln 420 425 430 7 410 PRTMus musculus 7 Leu Cys Gly Leu Tyr His Glu Ala Gly Gln Gln Leu Gln ArgLeu Lys 1 5 10 15 Asp Gln Leu Ala Ala Arg Asp Ala Leu Ile Ala Ser LeuArg Thr Arg 20 25 30 Leu Ala Ala Leu Glu Gly His Thr Ala Pro Ser Leu ValAsp Ala Leu 35 40 45 Leu Asp Gln Val Glu Arg Phe Arg Glu Gln Leu Arg ArgGln Glu Glu 50 55 60 Gly Ala Ser Glu Thr Gln Leu Arg Gln Glu Val Glu ArgLeu Thr Glu 65 70 75 80 Arg Leu Glu Glu Lys Glu Arg Glu Met Gln Gln LeuMet Ser Gln Pro 85 90 95 Gln His Glu Gln Glu Lys Glu Val Val Leu Leu ArgArg Ser Val Ala 100 105 110 Glu Lys Glu Lys Ala Arg Ala Ala Ser Asp ValLeu Cys Arg Ser Leu 115 120 125 Ala Asp Glu Thr His Gln Leu Arg Arg ThrLeu Ala Ala Thr Ala His 130 135 140 Met Cys Gln His Leu Ala Lys Cys LeuAsp Glu Arg Gln Cys Ala Gln 145 150 155 160 Gly Asp Ala Gly Glu Lys SerPro Ala Glu Leu Glu Gln Thr Ser Ser 165 170 175 Asp Ala Ser Gly Gln SerVal Ile Lys Lys Leu Gln Glu Glu Asn Arg 180 185 190 Leu Leu Lys Gln LysVal Thr His Val Glu Asp Leu Asn Ala Lys Trp 195 200 205 Gln Arg Tyr AspAla Ser Arg Asp Glu Tyr Val Lys Gly Leu His Ala 210 215 220 Gln Leu LysArg Arg Gln Val Pro Leu Glu Pro Glu Leu Met Lys Lys 225 230 235 240 GluIle Ser Arg Leu Asn Arg Gln Leu Glu Glu Lys Ile Ser Asp Cys 245 250 255Ala Glu Ala Asn Gln Glu Leu Thr Ala Met Arg Met Ser Arg Asp Thr 260 265270 Ala Leu Glu Arg Val Gln Met Leu Glu Gln Gln Ile Leu Ala Tyr Lys 275280 285 Asp Asp Phe Lys Ser Glu Arg Ala Asp Arg Glu Arg Ala His Ser Arg290 295 300 Ile Gln Glu Leu Glu Glu Lys Ile Met Ser Leu Met Tyr Gln ValSer 305 310 315 320 Gln Arg Gln Asp Ser Arg Glu Pro Gly Pro Cys Arg IleHis Thr Gly 325 330 335 Asn Lys Thr Ala Lys Tyr Leu Glu Met Asp Ala LeuGlu His Val Thr 340 345 350 Pro Gly Gly Trp Arg Pro Glu Ser Arg Ser GlnGln Met Glu Pro Ser 355 360 365 Ala Glu Gly Gly His Val Cys Thr Ala GlnArg Gly Gln Gly Asp Leu 370 375 380 Gln Cys Pro His Cys Leu Arg Cys PheSer Asp Glu Gln Gly Glu Ala 385 390 395 400 Phe Leu Arg His Leu Ser GluCys Cys Gln 405 410 8 19 PRT Artificial Sequence Description ofArtificial Sequence consensus amino acid sequence 1 8 Glu Xaa Xaa XaaLys Glu Ile Xaa Arg Leu Asn Xaa Xaa Leu Glu Glu 1 5 10 15 Xaa Xaa Ser 921 PRT Artificial Sequence Description of Artificial Sequence consensusamino acid sequence 2 9 Leu Xaa Gln Gln Xaa Xaa Xaa Xaa Xaa Xaa Asp PheXaa Xaa Glu Arg 1 5 10 15 Xaa Asp Arg Glu Arg 20 10 228 PRT Mus musculus10 Arg Gln Arg Glu Tyr Gln Glu Lys Glu Ile Gln Arg Leu Asn Lys Ala 1 510 15 Leu Glu Glu Ala Leu Ser Ile Gln Ala Ser Pro Ser Ser Pro Pro Ala 2025 30 Ala Phe Gly Ser Pro Glu Gly Val Gly Gly His Leu Arg Lys Gln Glu 3540 45 Leu Val Thr Gln Asn Glu Leu Leu Lys Gln Gln Val Lys Ile Phe Glu 5055 60 Glu Asp Phe Gln Arg Glu Arg Ser Asp Arg Glu Arg Met Asn Glu Glu 6570 75 80 Lys Glu Glu Leu Lys Lys Gln Val Glu Lys Leu Gln Ala Gln Val Thr85 90 95 Leu Thr Asn Ala Gln Leu Lys Thr Leu Lys Glu Glu Glu Lys Ala Lys100 105 110 Glu Ala Leu Lys Gln Gln Lys Arg Lys Ala Lys Ala Ser Gly GluArg 115 120 125 Tyr His Met Glu Pro His Pro Glu His Val Cys Gly Ala TyrPro Tyr 130 135 140 Ala Tyr Pro Pro Met Pro Ala Met Val Pro His His AlaTyr Lys Asp 145 150 155 160 Trp Ser Gln Ile Arg Tyr Pro Pro Pro Pro ValPro Met Glu His Pro 165 170 175 Pro Pro His Pro Asn Ser Arg Leu Phe HisLeu Pro Glu Tyr Thr Trp 180 185 190 Arg Pro Pro Cys Ala Gly Ile Arg AsnGln Ser Ser Gln Val Met Asp 195 200 205 Pro Pro Pro Asp Arg Pro Ala GluPro Glu Ser Ala Asp Asn Asp Cys 210 215 220 Asp Gly Pro Gln 225 11 50DNA Artificial Sequence Primer 11 gaataccagg aggcgcagat ccagcggctcaataaagctt tggaggaggc 50 12 35 DNA Artificial Sequence Primer 12gttgctgaaa gaggacgtca aaatctttga agagg 35 13 39 DNA Artificial SequencePrimer 13 gcaggtaaaa atctttgaag agaatgccca gagggaacg 39 14 62 DNAArtificial Sequence Primer 14 gcaggtaaaa atctttgaag aggacttccagagggaacgg agtgatgcgc aacgcatgcc 60 cg 62 15 19 PRT Artificial SequenceDescription of Artificial Sequence mutant ABIN-MUT1 15 Glu Tyr Gln GluAla Gln Ile Gln Arg Leu Asn Lys Ala Leu Glu Glu 1 5 10 15 Ala Leu Ser 1621 PRT Artificial Sequence Description of Artificial Sequence mutantABIN-MUT2 16 Leu Lys Glu Glu Val Lys Ile Phe Glu Glu Asp Phe Gln Arg GluArg 1 5 10 15 Ser Asp Arg Glu Arg 20 17 21 PRT Artificial SequenceDescription of Artificial Sequence mutant ABIN-MUT3 17 Leu Lys Gln GlnVal Lys Ile Phe Glu Glu Asn Ala Gln Arg Glu Arg 1 5 10 15 Ser Asp ArgGlu Arg 20 18 21 PRT Artificial Sequence Description of ArtificialSequence mutant ABIN-MUT4 18 Leu Lys Gln Gln Val Lys Ile Phe Glu Glu AspPhe Gln Arg Glu Arg 1 5 10 15 Ser Asp Ala Gln Arg 20 19 594 PRT Musmusculus 19 Met Glu Ala Ser Arg Leu Arg Gln Lys Ala Glu Glu Leu Val LysAsp 1 5 10 15 Ser Glu Leu Ser Pro Pro Thr Ser Ala Pro Ser Leu Val SerPhe Asp 20 25 30 Asp Leu Ala Glu Leu Thr Gly Gln Asp Thr Lys Val Gln ValHis Pro 35 40 45 Ala Thr Ser Thr Ala Ala Thr Thr Thr Ala Thr Ala Thr ThrGly Asn 50 55 60 Ser Met Glu Lys Pro Glu Pro Ala Ser Lys Ser Pro Ser AsnGly Ala 65 70 75 80 Ser Ser Asp Phe Glu Val Val Pro Thr Glu Glu Gln AsnSer Pro Glu 85 90 95 Thr Gly Ser His Pro Thr Asn Met Met Asp Leu Gly ProPro Pro Pro 100 105 110 Glu Asp Ser Asn Leu Lys Leu His Leu Gln Arg LeuGlu Thr Thr Leu 115 120 125 Ser Val Cys Ala Glu Glu Pro Asp His Ser GlnLeu Phe Thr His Leu 130 135 140 Gly Arg Met Ala Leu Glu Phe Asn Arg LeuAla Ser Lys Val His Lys 145 150 155 160 Asn Glu Gln Arg Thr Ser Ile LeuGln Thr Leu Cys Glu Gln Leu Arg 165 170 175 Gln Glu Asn Glu Ala Leu LysAla Lys Leu Asp Lys Gly Leu Glu Gln 180 185 190 Arg Asp Leu Ala Ala GluArg Leu Arg Glu Glu Asn Thr Glu Leu Lys 195 200 205 Lys Leu Leu Met AsnSer Ser Cys Lys Glu Gly Leu Cys Gly Gln Pro 210 215 220 Ser Ser Pro LysPro Glu Gly Ala Gly Lys Lys Gly Val Ala Gly Gln 225 230 235 240 Gln GlnAla Ser Val Met Ala Ser Lys Val Pro Glu Ala Gly Ala Phe 245 250 255 GlyAla Ala Glu Lys Lys Val Lys Leu Leu Glu Gln Gln Arg Met Glu 260 265 270Leu Leu Glu Val Asn Lys Gln Trp Asp Gln His Phe Arg Ser Met Lys 275 280285 Gln Gln Tyr Glu Gln Lys Ile Thr Glu Leu Arg Gln Lys Leu Val Asp 290295 300 Leu Gln Lys Gln Val Thr Glu Leu Glu Ala Glu Arg Glu Gln Lys Gln305 310 315 320 Arg Asp Phe Asp Arg Lys Leu Leu Leu Ala Lys Ser Lys IleGlu Met 325 330 335 Glu Glu Thr Asp Lys Glu Gln Leu Thr Ala Glu Ala LysGlu Leu Arg 340 345 350 Gln Lys Val Arg Tyr Leu Gln Asp Gln Leu Ser ProLeu Thr Arg Gln 355 360 365 Arg Glu Tyr Gln Glu Lys Glu Ile Gln Arg LeuAsn Lys Ala Leu Glu 370 375 380 Glu Ala Leu Ser Ile Gln Ala Ser Pro SerSer Pro Pro Ala Ala Phe 385 390 395 400 Gly Ser Pro Glu Gly Val Gly GlyHis Leu Arg Lys Gln Glu Leu Val 405 410 415 Thr Gln Asn Glu Leu Leu LysGln Gln Val Lys Ile Phe Glu Glu Asp 420 425 430 Phe Gln Arg Glu Arg SerAsp Arg Glu Arg Met Asn Glu Glu Lys Glu 435 440 445 Glu Leu Lys Lys GlnVal Glu Lys Leu Gln Ala Gln Val Thr Leu Thr 450 455 460 Asn Ala Gln LeuLys Thr Leu Lys Glu Glu Glu Lys Ala Lys Glu Ala 465 470 475 480 Leu LysGln Gln Lys Arg Lys Ala Lys Ala Ser Gly Glu Arg Tyr His 485 490 495 MetGlu Pro His Pro Glu His Val Cys Gly Ala Tyr Pro Tyr Ala Tyr 500 505 510Pro Pro Met Pro Ala Met Val Pro His His Ala Tyr Lys Asp Trp Ser 515 520525 Gln Ile Arg Tyr Pro Pro Pro Pro Val Pro Met Glu His Pro Pro Pro 530535 540 His Pro Asn Ser Arg Leu Phe His Leu Pro Glu Tyr Thr Trp Arg Pro545 550 555 560 Pro Cys Ala Gly Ile Arg Asn Gln Ser Ser Gln Val Met AspPro Pro 565 570 575 Pro Asp Arg Pro Ala Glu Pro Glu Ser Ala Asp Asn AspCys Asp Gly 580 585 590 Pro Gln

What is claimed is:
 1. A method of screening a compound for its abilityto interact with A20 interacting proteins, said method comprising:combining a sample, comprising a compound, to be screened with a proteinof an NF-κB related pathway in an assay; conducting the assay underconditions which permit an interaction of said protein of the NF-κBrelated pathway with the compound of said sample to be screened;detecting an interaction between said protein of the NF-κB relatedpathway and the compound; and identifying the compound in said samplethat interacts with said protein of the NF-κB related pathway in saidassay.
 2. The method according to claim 1, wherein said protein of theNF-κB related pathway comprises an A20 protein.
 3. The method accordingto claim 1, wherein said protein of the NF-κB related pathway has asequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:19, SEQ ID NO:2 and SEQ ID NO:5.
 4. The method according toclaim 3, further comprising combining said sample to be screened and anA20 polypeptide with said protein of the NF-κB related pathway in anassay.
 5. The method according to claim 4, further comprising assayingsaid sample to be screened for suppression of an interaction betweensaid protein of the NF-κB related pathway and said A20 polypeptide. 6.The method according to claim 4, further comprising assaying said sampleto be screened for activation of an interaction between said protein ofthe NF-κB related pathway and said A20 polypeptide.
 7. The methodaccording to claim 3, wherein preparing said assay comprises preparingan assay selected from the group consisting of a two-hybrid assay and aco-immunoprecipitation assay.
 8. The method according to claim 3,further comprising assaying said compound for activation or suppressionof ABIN dependent NF-κB inhibition.
 9. The method according to claim 8,further comprising assaying said compound for activation or suppressionof ABIN dependent NF-κB inhibition of TNF or IL-1 induced activation ofNF-κB inhibition.
 10. A method of screening a compound for its abilityto interact with A20 interacting proteins, said method comprising:preparing a read out system capable of detecting an interaction betweena protein of the NF-κB related pathway and a compound in a sample to bescreened; combining said sample to be screened with said protein of theNF-κB related pathway and said read out system in a reaction mixture;maintaining said reaction mixture under conditions that permitinteraction of said protein of the NF-κB related pathway with said readout system; and identifying a compound in said sample which interactswith said protein of the NF-κB related pathway in said read out system.11. The method according to claim 10, wherein said protein of the NF-κBpathway comprises A20.
 12. The method according to claim 10, whereinpreparing said read out system comprises preparing a two-hybrid assay.13. The method according to claim 10, wherein said protein of the NF-κBpathway comprises a sequence selected from the group consisting of SEQID NO:8, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:2 and SEQ ID NO:5.
 14. Themethod according to claim 13, further comprising identifying saidcompound in said sample that suppresses an interaction between saidprotein of the NF-κB pathway and A20.
 15. The method according to claim13, further comprising identifying said compound in said sample thatactivates an interaction between said protein of the NF-κB pathway andA20.
 16. The method according to claim 13, further comprising assayingthe compound for activation or suppression of NF-κB inhibition.
 17. Themethod according to claim 13, further comprising assaying the compoundfor activation or suppression of TNF or IL-1 induced activation ofNF-κB.